Cancer antigen targets and uses thereof

ABSTRACT

The presently disclosed subject matter provides methods and compositions for treating myeloid disorders (e.g., acute myeloid leukemia (AML)). It relates to immunoresponsive cells bearing antigen recognizing receptors (e.g., chimeric antigen receptors (CARs)) targeting AML-specific antigens.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of International Patent ApplicationNo. PCT/US17/045,632, filed Aug. 4, 2017, which claims priority to U.S.Provisional Application No. 62/371,199 filed on Aug. 4, 2016, thecontents of each of which are hereby incorporated by reference in theirentirety herein, and to each of which priority is claimed.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Apr. 30, 2018. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as0727340681SL.txt, is 38,303 bytes and was created on Apr. 30, 2018. TheSequence Listing, electronically filed herewith, does not extend beyondthe scope of the specification and thus does not contain new matter.

INTRODUCTION

The presently disclosed subject matter provides methods and compositionsfor treating cancer (e.g., acute myeloid leukemia (AML)). It relates toimmunoresponsive cells comprising antigen recognizing receptors (e.g.,chimeric antigen receptors (CARs)) targeting AML-specific antigens.

BACKGROUND OF THE INVENTION

Adoptive T cell therapies using CARs to redirect the specificity andfunction of T lymphocytes have demonstrated great efficacy in patientswith lymphoid malignancies, in particular acute lymphoblastic leukemia(ALL) (Sadelain, 2015). This therapeutic modality induces completeremissions in subjects with CD19⁺ malignancies for whom chemotherapieshave led to drug resistance and tumor progression. “Cancerimmunotherapy”, including CAR therapy, was proclaimed a scientificbreakthrough in 2013 (Couzin-Frankel, 2013). The success of CD19 CARtherapy bodes well for tackling all hematological malignancies,including Acute Myeloid Leukemia (AML), which affects over one quartermillion adults annually worldwide.

AML is the most common acute leukemia in adults. The standard inductionchemotherapy regimens have not changed substantially over the past 40years (Pulte et al., 2008) and the overall survival remains very poor.Frequent recurring abnormalities involving genes coding for epigeneticmodifiers have been identified. These epigenetic abnormalities includemutations in DNA-methylation related genes (DNMT3A, IDH1/2 in 44% ofpatients) (Cancer Genome Atlas Research, 2013), which also represent keyinitiating events in leukemogenesis (Shlush et al., 2014b). Molecularlytargeted therapies, such as IDH1/IDH2 and FLT3 inhibitors, are currentlyin clinical evaluation. The genetic engineering of T cells with CARsmediating antigen recognition, T cell activation, and co-stimulation, isattractive in that it rests on T cell-mediated cytotoxicity, withoutcausing reprogramming or metabolic changes. Unlike the physiological Tcell receptor, which engages HLA-peptide complexes, CARs bind to nativecell surface molecules and do not require any antigen processing or HLAexpression for tumor recognition. CARs therefore can recognize targetantigens on any HLA background or even on target tumor cells that havedown-regulated HLA expression or proteasomal antigen processing, twomechanisms that contribute to tumor escape from TCR-mediated immunity(Zhou and Levitsky, 2012). The target must, however, be found on thetumor cell surface.

The development of CAR therapy for AML is hampered by the lack ofsuitable targets. Identifying appropriate CAR targets is important toachieving complete tumor eradication, as is avoiding damage to normaltissues that express the same target antigen (“on-target, off-tumoreffect”). So far the search for suitable CAR targets has been limitedfor several reasons. For example, the major focus of researchers hasbeen restricted to the relative expression of potential targets incancer cells compared to normal counterparts. While it is true that anideal CAR target should be expressed in most if not all tumor cells,enabling efficient targeting by CAR⁺ T cells, it is also very importantto consider a whole body picture. For safe discrimination of targetcells by CAR⁺ T cells, an ideal tumor target should not be expressed onany normal tissue/organ of the whole body, including closely relatednormal counterparts (i.e., CD34⁺ hematopoietic stem/progenitor cells(HSPCs) and CD34⁺CD38⁻ hematopoietic stem cells (HSCs) in this case) andhealthy T cells, which mediate CAR therapy (to avoid fratricidekilling). If present on normal cells, the target should be at leastrestricted to non-vital tissues (as is the case of CD19, which is onlyfound in the normal B cell lineage). CD19 is the poster child of CARtherapy, found on most B lineage lymphomas and leukemias (LeBien andTedder, 2008). Thus, CD19 CAR therapy is expected to induce a B cellaplasia, as was observed in murine models (Davila et al., 2013; Pegramet al., 2012) and later in leukemia and lymphoma patients. Most targetsof CAR T cells have shared expression on normal tissues and some degreeof “on-target/off-tumor” toxicity occurred through engagement of targetantigen on nonpathogenic tissues (Curran et al., 2012). The severity ofreported events has ranged from manageable lineage depletion (B-cellaplasia) to severe toxicity (death). “On-target/off-tumor” recognitionis predictably seen in a variety of organ systems, includinggastrointestinal, hematologic, and pulmonary. One of the earliest trialsutilizing a carboxyanhydrase-IX-specific CAR T cell for renal cellcarcinoma resulted in the development of cholestasis due to expressionof carboxyanhydrase-IX on bile duct epithelium (Lamers et al., 2013;Lamers et al., 2006). Targeting of carcinoembryonic antigen by CAR Tcells in patients with colon cancer resulted in severe, albeittransient, colitis due to antigen recognition of normal colonic tissue(Parkhurst et al., 2011). Finally, in a fatal example of“on-target/off-tumor” recognition, a patient treated with CAR T cellsspecific for the cancer-associated antigen HER-2/neu developed rapidrespiratory failure, multi-organ dysfunction, and subsequent deathattributed to reactivity against pulmonary tissue expression ofHER-2/neu (Morgan et al., 2010).

In the case of AML, multiple genetic clones exist at diagnosis andcontribute to relapse, creating a complex and heterogeneous target proneto conventional and targeted therapies. The ideal CAR target should beexpressed on the driver leukemic clones, which survive chemotherapy andpersist during remission, to enable AML eradication by CAR⁺ T cells.

Furthermore, studies have shown that there exists a poor correlationbetween mRNA expression and protein abundance (Haider and Pal, 2013). Sofar the search for CAR targets relied mostly on the measurement oftranscriptomic profiles through techniques such as microarray andRNA-seq. However, the recent advancement in proteomic studies withMass-Spectometry and refined techniques in isolation of plasma cellmembrane offers additional sources of information to probe the cancersurfaceome and the integration of the two approaches is ideal.

Four CAR targets to AML have been reported in the literature. The first,Lewis (Le)-Y, a difucosylated carbohydrate antigen, was targeted in aphase I study of four patients with relapsed AML. Infusion of secondgeneration CD28-based CARs resulted in stable/transient remission ofthree patients, who ultimately progressed, despite T cell persistence(Ritchie et al., 2013). Regarding the second, CD123, the high-affinityinterleukin-3 receptor α-chain; a partial remission was induced in apatient with FLT3-ITD⁺ AML treated with a third generationCD123-CD28/CD137/CD27/CD3z/iCaps9 CAR (Yi Luo, 2015). Preclinicalstudies resulted in significant myeloablation (Gill et al., 2014). Thethird, CD33, is a myeloid-specific sialic acid-binding receptor which isalso targeted by gentuzumab ozogamicin (GO) (Administration, 2010), withdemonstrated survival benefit (Hills et al., 2014; Ravandi et al.,2012). Preclinical activity of CD33 CAR⁺ CIK cells resulted in slowingdisease progression (Pizzitola et al., 2014) and CD33 CAR⁺ T showedsignificant effector functions in vitro and in vivo with reduction ofmyeloid progenitors (Kenderian et al., 2015). One AML patient wastreated with CD33 CAR T cells at the Chinese PLA General Hospital,showing transient efficacy and mild fluctuations in bilirubin (Wang etal., 2015) and a clinical trial is registered as NCT01864902. Thefourth, folate receptor β, is a myeloid-lineage antigen (Lynn et al.,2016; Lynn et al., 2015).

However, none of these meet the criteria of an ideal CAR target.Accordingly, there are needs for novel therapeutic strategies to designCARs targeting antigens that are highly expressed in AML cells andlimited expression in normal tissues for treating AML, and forstrategies capable of inducing potent cancer eradication with minimaltoxicity and immunogenicity.

SUMMARY OF THE INVENTION

The presently disclosed subject matter provides immunoresponsive cells(e.g., T cells, Tumor Infiltrating Lymphocytes, Natural Killer (NK)cells, cytotoxic T lymphocytes (CTLs), Natural Killer T (NKT) cells orregulatory T cells), comprising an antigen recognizing receptor (e.g.,CAR or TCR) that binds to an antigen, which is an effective therapeuticagent against a myeloid disorder, for example, AML. In certainnon-limiting embodiments, an immunoresponsive cell, such as animmunoresponsive T cell or NK cell, can be engineered to express acombination of two or more CAR, TCR, and/or co-stimulatory receptor(“CCR”) that bind to one or more antigen to achieve activation andstimulation of the immunoresponsive T cell or NK cell. In certainnon-limiting embodiments, an immunoresponsive T cell can be engineeredto express a combination of CAR, TCR, and/or CCR that bind to differentantigens to achieve activation and stimulation of the immunoresponsive Tcell. In certain non-limiting embodiments, the one or more antigen isselected from the group consisting of EMR2, CD33, IL10RB, PLXNC1,PIEZO1, CD300LF, CPM, ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6,ENG, SIRPB1, MRP1, ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5,TFR2, KCNN4, LILRB4, LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5,PTPRJ, SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2,P2RY13, LILRB2, EMB, CD96, LILRB3, LILRA6, LILRA2, and SLC19A1. Incertain embodiments, the one or more antigen is selected from the groupconsisting of LTB4R, EMR2, CD33, MYADM, PIEZO1, SIRPB1, SLC9A1, KCNN4,ENG, ITGA5, and CD70. In certain embodiments, the one or more antigen isselected from the group consisting of LTB4R, EMR2, MYADM and PIEZO1. Incertain embodiments, the one or more antigen is selected from the groupconsisting of CD82, TNFRSF1B, EMR2, ITGB5, CCR1, CD96, PTPRJ, CD70 andLILRB2. In certain embodiments, the one or more antigen is selected fromthe group consisting of TNFRSF1B, EMR2, CCR1, CD96, CD70 and LILRB2. Incertain embodiments, the one or more antigen is selected from the groupconsisting of EMR2, CCR1, CD70 and LILRB2. In certain non-limitingembodiments, at least one of the one or more antigen is EMR2.

The presently disclosed subject matter further provides animmunoresponsive cell that comprises (i) an antigen recognizing receptor(e.g. CAR or TCR) that binds to a first antigen, wherein binding of theantigen recognizing receptor to the first antigen is capable ofactivating the immunoresponsive cell; and (ii) a CCR that binds to asecond antigen, wherein binding of the CCR to the second antigen iscapable of stimulating the immunoresponsive cell, wherein each of thefirst antigen and the second antigen is selected from the groupconsisting of EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF, CPM, ITFG3,TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1, MRP1, ITGA5,SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4, LILRB4,LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ, SLC30A1, EMC10,TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13, LILRB2, EMB,CD96, LILRB3, LILRA6, LILRA2, and SLC19A1, and the first antigen and thesecond antigen are different. In certain embodiments, the first antigenand the second antigen are a combination selected from the groupconsisting of LTB4R and EMR2, LTB4R and CD33, LTB4R and ENG, LTB4R andMYADM, LTB4R and PIEZO1, LTB4R and SIRPB1, LTB4R and SLC9A1, LTB4R andITGA5, LTB4R and CD70, LTB4R and KCNN4. EMR2 and CD33, EMR2 and ENG,EMR2 and MYADM, EMR2 and PIEZO1, EMR2 and SIRPB1, EMR2 and SLC9A1, EMR2and ITGA5, EMR2 and CD70, EMR2 and KCNN4, CD33 and ENG, CD33 and MYADM,CD33 and PIEZO1, CD33 and SIRPB1, CD33 and SLC9A1, CD33 and ITGA5, CD33and CD70, CD33 and KCNN4, ENG and MYADM, ENG and PIEZO1, ENG and SIRPB1,ENG and SLC9A1, ENG and ITGA5, ENG and CD70, ENG and KCNN4, MYADM andPIEZO1, MYADM and SIRPB1, MYADM and SLC9A1, MYADM and ITGA5, MYADM andCD70, MYADM and KCNN4, PIEZO1 and SIRPB1, PIEZO1 and SLC9A1, PIEZO1 andITGA5, PIEZO1 and CD70, PIEZO1 and KCNN4, SIRPB1 and SLC9A1, SIRPB1 andITGA5, SIRPB1 and CD70, SIRPB1 and KCNN4, SLC9A1 and ITGA5, SLC9A1 andCD70, SLC9A1 and KCNN4, ITGA5 and CD70, ITGA5 and KCNN4, CD70 and KCNN4,EMR2 and CD33, CCR1 and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A, EMR2and CLEC12A, EMR2 and CD96, CCR1 and CD33, CCR1 and CD96, CD70 andCLEC12A, CD70 and CD96, LILRB2 and CD33, LILRB2 and CD96, EMR2 and CD70.In certain embodiments, the first antigen and the second antigen are acombination selected from the group consisting of EMR2 and CD33, CCR1and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A, LTB4R1 and CD70, CD70and EMR2, and LTB4R1 and EMR2. In addition, in non-limiting embodiments,where the antigen recognizing receptor is a TCR, a target antigen can beWT1 or PRAME in addition to the aforementioned target antigens.

The presently disclosed subject matter further provides animmunoresponsive cell that comprises (i) a first antigen recognizingreceptor (e.g. CAR or TCR) that binds to a first antigen and (ii) asecond antigen recognizing receptor (e.g. CAR or TCR) that binds to asecond antigen, wherein the combination of both receptors binding totheir targets produces a therapeutic effect. In certain non-limitingembodiments, binding to only one target does not achieve a therapeuticeffect. For example, the first and second antigen recognizing receptorcan both be CARs; alternatively, the first antigen recognizing receptorcan be a CAR and the second antigen binding receptor can be a TCR, orthe first antigen recognizing receptor can be a TCR and the secondantigen recognizing receptor can be a CAR, or both antigen recognizingreceptors can be TCRs. Optionally, said immunoresponsive cell mayfurther comprise a third antigen targeting molecule, which may be a CAR,TCR, or CCR that recognizes a third antigen. In non-limitingembodiments, the first, second, and optional third antigen aredifferent. In non-limiting embodiments, each of the first antigen,second antigen and third antigen is selected from the group consistingof EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF, CPM, ITFG3, TTYH3,ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1, MRP1, ITGA5, SLC43A3,MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4, LILRB4, LTB4R, CD70,GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ, SLC30A1, EMC10, TNFRSF1B,CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13, LILRB2, EMB, CD96,LILRB3, LILRA6, LILRA2, and SLC19A1, and the first antigen and thesecond antigen are different. In certain embodiments, the first antigenand the second antigen are a combination selected from the groupconsisting of LTB4R and EMR2, LTB4R and CD33, LTB4R and ENG, LTB4R andMYADM, LTB4R and PIEZO1, LTB4R and SIRPB1, LTB4R and SLC9A1, LTB4R andITGA5, LTB4R and CD70, LTB4R and KCNN4. EMR2 and CD33, EMR2 and ENG,EMR2 and MYADM, EMR2 and PIEZO1, EMR2 and SIRPB1, EMR2 and SLC9A1, EMR2and ITGA5, EMR2 and CD70, EMR2 and KCNN4, CD33 and ENG, CD33 and MYADM,CD33 and PIEZO1, CD33 and SIRPB1, CD33 and SLC9A1, CD33 and ITGA5, CD33and CD70, CD33 and KCNN4, ENG and MYADM, ENG and PIEZO1, ENG and SIRPB1,ENG and SLC9A1, ENG and ITGA5, ENG and CD70, ENG and KCNN4, MYADM andPIEZO1, MYADM and SIRPB1, MYADM and SLC9A1, MYADM and ITGA5, MYADM andCD70, MYADM and KCNN4, PIEZO1 and SIRPB1, PIEZO1 and SLC9A1, PIEZO1 andITGA5, PIEZO1 and CD70, PIEZO1 and KCNN4, SIRPB1 and SLC9A1, SIRPB1 andITGA5, SIRPB1 and CD70, SIRPB1 and KCNN4, SLC9A1 and ITGA5, SLC9A1 andCD70, SLC9A1 and KCNN4, ITGA5 and CD70, ITGA5 and KCNN4, CD70 and KCNN4,EMR2 and CD33, CCR1 and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A, EMR2and CLEC12A, EMR2 and CD96, CCR1 and CD33, CCR1 and CD96, CD70 andCLEC12A, CD70 and CD96, LILRB2 and CD33, LILRB2 and CD96, EMR2 and CD70.In certain embodiments, the first antigen and the second antigen are acombination selected from the group consisting of EMR2 and CD33, CCR1and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A, LTB4R1 and CD70, CD70and EMR2, and LTB4R1 and EMR2 In certain non-limiting embodiments, thefirst antigen is EMR2. In addition, in non-limiting embodiments, wherean antigen recognizing receptor is a TCR, a target antigen can be WT1 orPRAME in addition to the aforementioned target antigens.

In certain embodiments, the aforementioned cell exhibits a greaterdegree of cytolytic activity against cells that are positive for boththe first antigen and the second antigen as compared to against cellsthat are singly positive for the first antigen.

In certain embodiments, the antigen recognizing receptor binds to thefirst antigen with a low binding affinity. In certain embodiments, theantigen recognizing receptor binds to the first antigen with adissociation constant (K_(d)) of 1×10⁻⁸ M or more. In certainembodiments, the antigen recognizing receptor binds to the first antigenwith a K_(d) of 5×10⁻⁸ M or more. In certain embodiments, the antigenrecognizing receptor binds to the first antigen with a K_(d) of 1×10⁻⁷ Mor more. In certain embodiments, the antigen recognizing receptor bindsto the first antigen with a K_(d) of 1×10⁻⁶ M or more. In certainembodiments, the antigen recognizing receptor (e.g. CAR or TCR) binds tothe first antigen with a binding affinity that is lower compared to thebinding affinity with which the second antigen recognizing receptor orCCR that binds to the second antigen. In certain embodiments, theantigen recognizing receptor (e.g. CAR or TCR) binds to the firstantigen with a low binding avidity. In certain embodiments, the antigenrecognizing receptor (e.g. CAR or TCR) binds to the first antigen at anepitope of low accessibility.

In certain embodiments, the CCR is recombinantly expressed. In certainembodiments, the CCR is expressed from a vector, or a selected locusfrom the genome of the immunoresponsive cell. In certain embodiments,the antigen recognizing receptor is a CAR. In certain embodiments, theCAR has a dissociation constant (K_(d)) of about 1×10⁻⁸ M to about1×10⁻⁶ M. In certain embodiments, the CCR has a K_(d) of about 1×10⁻⁹ Mto about 1×10⁻⁷ M.

Furthermore, the presently disclosed subject matter provides methods fortreating and/or preventing a myeloid disorder in a subject comprisingadministering an effective amount of aforementioned immunoresponsivecells. Non-limiting examples of myeloid disorder include myelodysplasticsyndromes, myeloproliferative neoplasms, chronic myelomonocyticleukemia, and acute myeloid leukemia (AML), acute myeloblastic leukemia,acute promyelocytic leukemia, acute myelomonocytic leukemia, chronicmyelocytic leukemia, and polycythemia vera. In certain embodiments, themyeloid disorder is AML. In certain embodiments, the method reduces oreradicates tumor burden in the subject and/or prolongs remission and/orprolongs survival.

The presently disclosed subject matter also provides methods of reducingtumor burden in a subject comprising administering an effective amountof presently disclosed immunoresponsive cells. In certain embodiments,the method reduces the number of tumor cells (e.g. leukemic cells). Incertain embodiments, the method prolongs survival of the subject.

The presently disclosed subject matter further provides methods forproducing an antigen-specific immunoresponsive cell. In certainembodiments, the method comprises introducing into the immunoresponsivecell a nucleic acid sequence encoding an antigen recognizing receptorthat binds to an antigen, wherein the antigen is selected from the groupconsisting of EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF, CPM, ITFG3,TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1, MRP1, ITGA5,SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4, LILRB4,LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ, SLC30A1, EMC10,TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13, LILRB2, EMB,CD96, LILRB3, LILRA6, LILRA2, and SLC19A1 and SLC19A1. In certainembodiments, the antigen is selected from the group consisting of LTB4R,EMR2, CD33, MYADM, PIEZO1, SIRPB1, SLC9A1, KCNN4, ENG, ITGA5, and CD70.In certain embodiments, the antigen is selected from the groupconsisting of LTB4R, EMR2, MYADM and PIEZO1.

In certain embodiments, the method for producing an antigen-specificimmunoresponsive cell comprises introducing into the immunoresponsivecell (a) a first nucleic acid sequence encoding an antigen recognizingreceptor (e.g., CAR or TCR) that binds to a first antigen, whereinbinding of the antigen recognizing receptor to the first antigen iscapable of activating the immunoresponsive cell, and (b) a secondnucleic acid sequence encoding a CCR that binds to a second antigen,wherein binding of the CCR to the second antigen is capable ofstimulating the immunoresponsive cell, wherein each of the first antigenand the second antigen is selected from the group consisting of EMR2,CD33, IL10RB, PLXNC1, PIEZO1, CD300LF, CPM, ITFG3, TTYH3, ITGA4, SLC9A1,MBOAT7, CD38, SLC6A6, ENG, SIRPB1, MRP1, ITGA5, SLC43A3, MYADM, ICAM1,SLC44A1, CCR1, SLC22A5, TFR2, KCNN4, LILRB4, LTB4R, CD70, GYPA, FCGR1A,CD123, CLEC12A, ITGB5, PTPRJ, SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX,CR1, DAGLB, SEMA4A, TLR2, P2RY13, LILRB2, EMB, CD96, LILRB3, LILRA6,LILRA2, and SLC19A1, and the first antigen and the second antigen aredifferent. In certain embodiments, the first antigen and the secondantigen are a combination selected from the group consisting of LTB4Rand EMR2, LTB4R and CD33, LTB4R and ENG, LTB4R and MYADM, LTB4R andPIEZO1, LTB4R and SIRPB1, LTB4R and SLC9A1, LTB4R and ITGA5, LTB4R andCD70, LTB4R and KCNN4. EMR2 and CD33, EMR2 and ENG, EMR2 and MYADM, EMR2and PIEZO1, EMR2 and SIRPB1, EMR2 and SLC9A1, EMR2 and ITGA5, EMR2 andCD70, EMR2 and KCNN4, CD33 and ENG, CD33 and MYADM, CD33 and PIEZO1,CD33 and SIRPB1, CD33 and SLC9A1, CD33 and ITGA5, CD33 and CD70, CD33and KCNN4, ENG and MYADM, ENG and PIEZO1, ENG and SIRPB1, ENG andSLC9A1, ENG and ITGA5, ENG and CD70, ENG and KCNN4, MYADM and PIEZO1,MYADM and SIRPB1, MYADM and SLC9A1, MYADM and ITGA5, MYADM and CD70,MYADM and KCNN4, PIEZO1 and SIRPB1, PIEZO1 and SLC9A1, PIEZO1 and ITGA5,PIEZO1 and CD70, PIEZO1 and KCNN4, SIRPB1 and SLC9A1, SIRPB1 and ITGA5,SIRPB1 and CD70, SIRPB1 and KCNN4, SLC9A1 and ITGA5, SLC9A1 and CD70,SLC9A1 and KCNN4, ITGA5 and CD70, ITGA5 and KCNN4, CD70 and KCNN4, EMR2and CD33, CCR1 and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A, EMR2 andCLEC12A, EMR2 and CD96, CCR1 and CD33, CCR1 and CD96, CD70 and CLEC12A,CD70 and CD96, LILRB2 and CD33, LILRB2 and CD96, and EMR2 and CD70. Incertain embodiments, the combination is selected from the groupconsisting of EMR2 and CD33, CCR1 and CLEC12A, CD70 and CD33, LILRB2 andCLEC12A, LTB4R1 and CD70, CD70 and EMR2, and LTB4R1 and EMR2.

In certain non-limiting embodiments, the presently disclosed subjectmatter provides a nucleic acid encoding an antigen recognizing receptorthat binds to an antigen. In certain embodiments, the antigen isselected from the group consisting of EMR2, CD33, IL10RB, PLXNC1,PIEZO1, CD300LF, CPM, ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6,ENG, SIRPB1, MRP1, ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5,TFR2, KCNN4, LILRB4, LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5,PTPRJ, SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2,P2RY13, LILRB2, EMB, CD96, LILRB3, LILRA6, LILRA2, and SLC19A1 andSLC19A1. The presently disclosed subject matter further provides avector comprising such nucleic acid. In certain non-limitingembodiments, the antigen recognizing receptor is a CAR. In certainembodiments, the vector is a retroviral vector.

The presently disclosed subject matter further provides pharmaceuticalcompositions comprising an effective amount of the presently disclosedimmunoresponsive cells and a pharmaceutically acceptable excipient. Incertain embodiments, the pharmaceutical composition is for treat orpreventing a myeloid disorder (e.g., AML).

Furthermore, the presently disclosed subject matter provides kits fortreating or preventing a myeloid disorder (e.g., AML), comprising one ormore presently disclosed immunoresponsive cells, or a presentlydisclosed nucleic acid. In certain embodiments, the kit furthercomprises written instructions for using the cell for treating and/orpreventing a myeloid disorder in a subject. The nucleic acid may encodemore than one antigen recognizing receptor, each may be operably linkedto a promoter which may be the same or different promoters. In certainembodiments, the kit further comprises written instructions for usingthe nucleic acids to produce a cell for treating and/or preventing amyeloid disorder in a subject.

The presently disclosed subject matter further provides an isolatedimmunoresponsive cell comprising an antigen recognizing receptor (e.g.,CAR or TCR) that binds to an antigen, wherein binding of the antigenrecognizing receptor to the antigen is capable of activating theimmunoreponsive cell, and wherein the antigen is selected from the groupconsisting of TMEM40, GNAZ, SLC6A16, PPP2R5B, TEX29, FKBP1B, KCNJ5,CAPN3, TNFRSF14, SPAG17, MMP25, NGFR, CLEC1A, OTOA, LRRN2, RHBDL3,HEPHL1, TSPEAR, TAS1R3, MBOAT1, MT-ND1, DARC, SH3PXD2A, BEST4, STON2,ACKR6, LRRTM2, STC1, SLC16A6, CDHR1, MYADML2, PNPLA3, PSD2, SLC25A41,SUSD2, KCND1, HILPDA, TMEM145, DFNB31, PPFIA4, NLGN3, FAM186B, KCNV2,SCN11A, ABCG2, ANO9, GAS2, ASIC3, B3GNT4, TMEM59L, SLC25A36, FRMD5,COL15A1, ZDHHC11, ITGA8, PEAR1, ASPRV1, LOXL4, TRIM55, KIF19, LPAR2,CNIH2, FLRT1, RNF183, RDH16, CADM3, C3orf35, GDPD3, TMPRSS5, SEC31B,AGER, ADAMTS13, IL20RB, WNT4, LRRC37A3, SCNN1D, TMEM89, EXOC3L4,ATP6VOA4, CHST3, NPAS2, IGFBP3, ADRAID, RNF173, CEACAM6, MANSC1, ELOVL6,LEPR, SUN3, HOOK1, CCDC155, TMEM27, GABRB2, EPHA4, CDH13, AQP2, KCNK13,KIF26B, HTR2A, SLC44A3, ILDR1, CYP4F11, SLC8A3, GPR153, SLCO2B1, SCIN,SCN2A, IL23R, ALS2, GNA14, TMEFF2, EXTL3, PDE3A, MFAP3L, SLC34A3,TACSTD2, ITGB8, LAX1, SLC45A3, SYNC, PLXNA4, ADORA3, SIGLEC11, RYR2,LRRC8E, DGKI, COLEC12, and CX3CR1.

The presently disclosed subject matter further provides an isolatedimmunoresponsive cell comprising: (a) an antigen recognizing receptorthat binds to a first antigen, wherein binding of the antigenrecognizing receptor to the first antigen is capable of activating theimmunoresponsive cell, and (b) a chimeric co-stimulating receptor (CCR)that binds to a second antigen, wherein binding of the CCR to the secondantigen is capable of stimulating the immunoresponsive cell, whereineach of the first antigen and the second antigen is selected from thegroup consisting of TMEM40, GNAZ, SLC6A16, PPP2R5B, TEX29, FKBP1B,KCNJ5, CAPN3, TNFRSF14, SPAG17, MMP25, NGFR, CLEC1A, OTOA, LRRN2,RHBDL3, HEPHL1, TSPEAR, TAS1R3, MBOAT1, MT-ND1, DARC, SH3PXD2A, BEST4,STON2, ACKR6, LRRTM2, STC1, SLC16A6, CDHR1, MYADML2, PNPLA3, PSD2,SLC25A41, SUSD2, KCND1, HILPDA, TMEM145, DFNB31, PPFIA4, NLGN3, FAM186B,KCNV2, SCN11A, ABCG2, ANO9, GAS2, ASIC3, B3GNT4, TMEM59L, SLC25A36,FRMD5, COL15A1, ZDHHC11, ITGA8, PEAR1, ASPRV1, LOXL4, TRIM55, KIF19,LPAR2, CNIH2, FLRT1, RNF183, RDH16, CADM3, C3orf35, GDPD3, TMPRSS5,SEC31B, AGER, ADAMTS13, IL20RB, WNT4, LRRC37A3, SCNN1D, TMEM89, EXOC3L4,ATP6VOA4, CHST3, NPAS2, IGFBP3, ADRAID, RNF173, CEACAM6, MANSC1, ELOVL6,LEPR, SUN3, HOOK1, CCDC155, TMEM27, GABRB2, EPHA4, CDH13, AQP2, KCNK13,KIF26B, HTR2A, SLC44A3, ILDR1, CYP4F11, SLC8A3, GPR153, SLCO2B1, SCIN,SCN2A, IL23R, ALS2, GNA14, TMEFF2, EXTL3, PDE3A, MFAP3L, SLC34A3,TACSTD2, ITGB8, LAX1, SLC45A3, SYNC, PLXNA4, ADORA3, SIGLEC11, RYR2,LRRC8E, DGKI, COLEC12, and CX3CR1, and the first antigen and the secondantigen are different.

In various embodiments of any of the aspects delineated herein, theantigen recognizing receptor is a T cell receptor (TCR) or chimericantigen receptor (CAR). In various embodiments of any of the aspectsdelineated herein, the antigen recognizing receptor is exogenous orendogenous. In various embodiments of any of the aspects delineatedherein, the antigen recognizing receptor is recombinantly expressed. Invarious embodiments of any of the aspects delineated herein, the antigenrecognizing receptor is expressed from a vector. The CAR can comprise anintracellular signaling domain. In various embodiments of any of theaspects delineated herein, the intracellular signaling domain is theCD3ζ-chain, CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB,CD28 signaling domain, a portion thereof, or combinations thereof. Incertain non-limiting embodiments, the antigen recognizing receptor is aCAR comprising at least a portion of CD28, 4-1BB, and/or CD3ζ-chain,together with an antigen binding portion. In certain non-limitingembodiments, the antigen recognizing receptor is a CAR described in Kohnet al., 2011, Molecular Ther. 19(3):432-438, optionally where theantigen binding portion is substituted with amino acid sequence thatbinds to another tumor or pathogen antigen. In various embodiments, thecell expresses a recombinant or an endogenous antigen receptor that is1928z or 4H1128z.

In an additional aspect, the invention provides a method for treating orpreventing a myeloid disorder, comprising administering an effectiveamount of at least one antibody that binds to an antigen selected fromthe group consisting of EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF,CPM, ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1,MRP1, ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4,LILRB4, LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ,SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13,LILRB2, EMB, CD96, LILRB3, LILRA6, LILRA2, and SLC19A1. In certainembodiments, the antigen is selected from the group consisting of LTB4R,EMR2, CD33, MYADM, PIEZO1, SIRPB1, SLC9A1, KCNN4, ENG, ITGA5, and CD70.In certain embodiments, the antigen is selected from the groupconsisting of LTB4R, EMR2, MYADM and PIEZO1.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description, given by way of example but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings.

FIGS. 1A-1E depict strategy and results of the study to identify CARtargets in AML. FIG. 1A depicts the screening strategy and databasesinvolved in study. FIG. 1B depicts the algorithm to identify suitableCAR targets in AML. FIG. 1C depicts the 32 “Rank Selection” proteinsfrom step #3 of FIG. 1B. FIG. 1D depicts the 11 top candidates from step#4 of FIG. 1B. FIG. 1E depicts the combinatorial targeting strategy withimmunoresponsive cells expressing both a suboptimal CAR and a chimericco-stimulatory receptor (CCR) recognizing a second antigen.

FIGS. 2A-2E depict the results of antigen expression analyses in normaland malignant cells by flow-cytometry. FIG. 2A shows expression of 9candidates: LTB4R, EMR2, SLC9A1, MYADM, CD33, SLC6A6, KCNN4, PIEZO1, andSIRPB1. FIG. 2B shows 3 candidates, CD70, ENG, and ITGA5 with highexpression in T cells. FIG. 2C shows 13 candidates, CCR1, SLC22A5, TFR2,LILRB4, GYPA, FCGR1A, IL10RB, PLXNC1, CD300LF, MBOAT7, MRP1, SLC43A3,and SLC44A1, which have a non-homogenous expression in all AML cells.FIG. 2D shows 6 candidates, CPM, TTYH3, ITGA4, SLC19A1, CD38, ICAM1,which have high expression in normal HSCs. FIG. 2E is a summary of FIGS.2A-2D.

FIGS. 3A-3B depict the results of further screening of CAR targets inAML. FIG. 3A depicts the algorithm that identifies the 55 pairs of CARtargets. FIG. 3B depicts the flow cytometry results verifying expressionof LTB4R1 and EMR2 in normal and malignant cells.

FIG. 4 depicts the combinatorial approach. Using CD70-CCR and CD33-CARas example, the figure illustrates the vector design, the expression inT cells by flow-cytometry and the preliminary data on AML cells by thecytotoxic T lymphocyte (CTL) assay.

FIGS. 5A-5B depict the RNA-sequencing results from pre-leukemic stemcells expressing indicated mutant genes and wildtype controls.

FIG. 6 depicts the generation of a comprehensive dataset of surfacemolecule annotations. On the left side the data sources related tomalignant (AML) cells and on the right the data sources related tonormal cells. Orange boxes represent the information derived from eitherprevious studies (346) or in-house surface proteomics studies (4,862) ina panel of AML cell lines. Yellow boxes represent the data sourcesproviding information regarding subcellular localization. Green boxesrepresent three distinct published repositories of protein expressionlevels in several normal tissues and the platform in which data wasgenerated. Pink boxes represent RNA data either from normal (right side)or AML cells (left side). The blue box represents the expression dataobtained by flow-cytometry in multiple distinct subsets of hematopoieticcells. The center grey box represents the combined annotationrepository.

FIGS. 7A-7B depicts an algorithm to identify candidates for CAR therapy.A) The algorithm shows the steps, which identify surface molecules inAML and molecules, which are overexpressed in AML compared to normalHSPCs; the quality control; the step, which identifies targets withminimal expression in a large panel of normal tissues andflow-cytometric analysis. Step descriptions color-coded relative to datasources in FIG. 6. Indicated to the right of each box, the number ofmolecules resulting from each analytical step. B) Heatmap showing theexpression profile of 24 selected candidates in a large panel of normaltissues as well as previously identified CAR targets in AML and CD19.*Only PDB distinguishes between CD44 and CD44v6, shown is an aggregateof CD44 isoforms. If one excluded high expression in the normal spleenfrom the analysis, both CD33 and CLEC12A would be included amongst thetop 24 CAR candidate targets (as illustrated in FIG. 14).

FIGS. 8A-8E depict the flow-cytometric analysis in primary AML patientsamples and normal hematopoietic cells. A) Percentage of cellsexpressing candidate antigens in AML bulk population with respect to 3CAR targets (CD123, CD33 and CLEC12A) in current clinical investigationsby flow-cytometry B) Percentage of cells expressing candidate antigensin Leukemic CD34+CD38+ stem cell population by flow-cytometry C)Percentage of cells expressing candidate antigens in normal bone marrowCD34+CD38−CD45RA-CD90+ HSCs and CD34+CD38+ progenitor cells byflow-cytometry. D) Percentage of cells expressing candidate antigens innormal CD3+ T cells at two time points (freshly purified and uponactivation) by flow-cytometry E) Summary expression levels of 4 toptargets in AML bulk population, LSCs, normal HSCs and T cells.****corresponds to P value <0.0001 by Student's t-test.

FIGS. 9A-9F depict the principles of pairwise analysis. A) An ideal pairshould not present overlapping expression in normal tissues. In theCAR/CAR approach, some low or moderate expression in normal tissues,albeit not optimal, may be tolerable depending on the tissues inquestion. In the CAR/CCR, T cells are more restricted to dual-antigenpositive tumor cells, thus relaxing the expression criteria for at leastone of the paired antigens. B) The expression of target pairs should bevery low in CD34+CD38− HSCs. C) The expression of two targets in a pairshould be very low in normal resting and activated (r/a) T cells. D)Each antigen in a pair may be differentially expressed in differentclones. The CAR/CCR approach requires expression of the CAR target. E)The pair should be expressed in leukemic stem cells. The CAR/CARapproach may pair an antigen expressed on most cells but not on LSCs. F)Co-targeting may prevent the emergence of clones that may downregulatethe expression of one antigen at a later time (t1).

FIGS. 10A-10D depict the combinatorial pairs of targets. A) 4combinatorial pairs, defined by evaluating the expression of tissuesites together. Criteria for vital tissues require at least one antigenin the pair to possess no detectable expression in any tissue. Criteriafor non-vital tissues permit tissue expression to exhibit “low”expression. B) Total percent expression of cells with either antigens inthe pair compared to expression levels of each antigen alone in primaryAML samples C) Levels of co-expression (intersection) of two targetscompared to total (union) expression levels. Data are represented asmean±standard deviation. D) Expression levels of the pairs in AML cellscompared to normal BM HSCs and T cells.

FIGS. 11A-11B depict the AML surface proteomics. A) Simplified diagramdepicting the principle of surface-specific proteomic analyses performedin a panel of AML cell lines B) Venn diagram comparing moleculesidentified by surface biotinylation in AML cells and reported surfacemarkers in AML.

FIGS. 12A-12B depict the calculation of distribution metrics in PDB andHPM datasets. A) PDB, the log 10 expression density, plotted with normalcurve overlay. Dashed black line placed at peak maximum and dashedpurple lines at one standard deviation above and below peak max. B) HPM,ordered log 10 expression. Dashed black line placed at peak maximum anddashed purple lines at one standard deviation above and below peak max.

FIG. 13 depicts the expression analysis of T cell targets with provenoff-tumor toxicity in normal tissues. Expression profile of ERBB2,targeted in breast cancer and related to toxicity in the lung (arrow);CAIX, targeted in renal cell carcinoma, and related to toxicity in thebiliary system (arrow at the site of gallbladder); CEACAM5, targeted incolon cancer and related to hemorrhagic colitis. High expression wasfound in the normal colon (arrow) as well as stomach and esophagus.Adoptively transferred T cells were found at these sites in treatedpatients.

FIG. 14 depicts the expression profile of CAR targets in normal tissues.Heatmap showing the expression profile of selected candidates with nohigh (3) expression in a large panel of normal tissues, except blood,bone marrow and spleen.

FIG. 15 depicts the scatter plots of the expressions of AML targets inpatient. First row shows 2 scatter plots of ADGRE2+CD33 pair from 2patients. Second row shows 2 scatter plots of CD70+CD33 pair from 2patients. Third row shows 2 scatter plots of CCR1+CLEC12A pair from 2patients and fourth row shows 2 scatter plots of LILRB2+CLEC12A pairfrom 2 patients. The presented data were acquired on different days andfrom different patients and analyzed in comparison to their specificcontrols.

FIG. 16 depicts ten combinatorial pairs with non-overlapping expressionin normal tissues.

DETAILED DESCRIPTION OF THE INVENTION

The presently disclosed subject matter provides cells, includinggenetically modified immunoresponsive cells (e.g., T cells, NaturalKiller (NK) cells, cytotoxic T lymphocytes (CTL) cells and regulatory Tcells) comprising one or more antigen recognizing receptor (e.g., TCR orCAR) that binds to an antigen of interest and can optionally furthercomprise a co-stimulatory receptor (CCR), and methods of using suchcells for treating and/or preventing myeloid disorders and otherpathologies where an antigen-specific immune response is desired. Thepresently disclosed subject matter is based, at least in part, on thediscovery of antigens specific to AML cells.

Malignant cells have developed a series of mechanisms to protectthemselves from immune recognition and elimination. The present approachprovides immunogenicity within the tumor microenvironment for tumoreradication, and represents a significant advance over conventionaladoptive T cell therapy. In certain non-limiting embodiments, itprovides an option of foregoing some or all ancillary treatments such asprior conditioning of the host with total body irradiation, high-dosechemotherapy, and/or postinfusion cytokine support.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

As used herein, the term “about” or “approximately” means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” can mean within 3 or more than 3 standarddeviations, per the practice in the art. Alternatively, “about” can meana range of up to 20%, preferably up to 10%, more preferably up to 5%,and more preferably still up to 1% of a given value. Alternatively,particularly with respect to biological systems or processes, the termcan mean within an order of magnitude, preferably within 5-fold, andmore preferably within 2-fold, of a value.

By “activates an immunoresponsive cell” is meant induction of signaltransduction or changes in protein expression in the cell resulting ininitiation of an immune response. For example, when CD3 Chains clusterin response to ligand binding and immunoreceptor tyrosine-basedinhibition motifs (ITAMs) a signal transduction cascade is produced. Incertain embodiments, when an endogenous TCR or an exogenous CAR bindsantigen, a formation of an immunological synapse occurs that includesclustering of many molecules near the bound receptor (e.g. CD4 or CD8,CD3γ/δ/ε/ζ, etc.) This clustering of membrane bound signaling moleculesallows for ITAM motifs contained within the CD3 chains to becomephosphorylated. This phosphorylation in turn initiates a T cellactivation pathway ultimately activating transcription factors, such asNF-κB and AP-1. These transcription factors induce global geneexpression of the T cell to increase IL-2 production for proliferationand expression of master regulator T cell proteins in order to initiatea T cell mediated immune response.

By “stimulates an immunoresponsive cell” is meant a signal that resultsin a robust and sustained immune response. In various embodiments, thisoccurs after immune cell (e.g., T-cell) activation or concomitantlymediated through receptors including, but not limited to, CD28, CD137(4-1BB), OX40, CD40 and ICOS. Without being bound to a particulartheory, receiving multiple stimulatory signals is important to mount arobust and long-term T cell mediated immune response. Without receivingthese stimulatory signals, T cells quickly become inhibited andunresponsive to antigen. While the effects of these co-stimulatorysignals vary and remain partially understood, they generally result inincreasing gene expression in order to generate long lived,proliferative, and anti-apoptotic T cells that robustly respond toantigen for complete and sustained eradication.

The term “antigen recognizing receptor” as used herein refers to areceptor that is capable of activating an immune cell (e.g., a T-cell)in response to antigen binding. Exemplary antigen recognizing receptorsmay be native or endogenous T cell receptors or chimeric antigenreceptors in which an antigen-binding domain is fused to anintracellular signaling domain capable of activating an immune cell(e.g., a T-cell).

As used herein, the term “antibody” means not only intact antibodymolecules, but also fragments of antibody molecules that retainimmunogen-binding ability. Such fragments are also well known in the artand are regularly employed both in vitro and in vivo. Accordingly, asused herein, the term “antibody” means not only intact immunoglobulinmolecules but also the well-known active fragments F(ab′)₂, and Fab.F(ab′)₂, and Fab fragments that lack the Fe fragment of intact antibody,clear more rapidly from the circulation, and may have less non-specifictissue binding of an intact antibody (Wahl et al., J Nucl. Med.24:316-325 (1983). The antibodies of the invention comprise whole nativeantibodies, bispecific antibodies; chimeric antibodies; Fab, Fab′,single chain V region fragments (scFv), fusion polypeptides, andunconventional antibodies.

As used herein, the term “single-chain variable fragment” or “scFv” is afusion protein of the variable regions of the heavy (VH) and lightchains (VL) of an immunoglobulin covalently linked to form a VH::VLheterodimer. The heavy (VH) and light chains (VL) are either joineddirectly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25amino acids), which connects the N-terminus of the VH with the Cterminus of the VL, or the C-terminus of the VH with theN-terminus ofthe VL. The linker is usually rich in glycine for flexibility, as wellas serine or threonine for solubility. Despite removal of the constantregions and the introduction of a linker, scFv proteins retain thespecificity of the original immunoglobulin. Single chain Fv polypeptideantibodies can be expressed from a nucleic acid including VH- and VLencoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci.USA, 85:5879-5883, 1988). See, also, U.S. Pat. Nos. 5,091,513, 5,132,405and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and20050196754. Antagonistic scFvs having inhibitory activity have beendescribed (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 200827(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 Aug. 12;Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., ThrombHaemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84;Moosmayer et al., Ther Immunol 1995 2 (10:31-40). Agonistic scFvs havingstimulatory activity have been described (see, e.g., Peter et al., JBioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 199715(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17 (5-6):427-55;Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

As used herein, the term “affinity” refers to a measure of bindingstrength. Without being bound to theory, affinity depends on thecloseness of stereochemical fit between antibody combining sites andantigen determinants, on the size of the area of contact between them,and on the distribution of charged and hydrophobic groups. Affinity alsoincludes the term “avidity,” which refers to the strength of theantigen-antibody bond after formation of reversible complexes. Methodsfor calculating the affinity of an antibody for an antigen are known inthe art, including use of binding experiments to calculate affinity.Antibody activity in functional assays (e.g., flow cytometry assay) isalso reflective of antibody affinity. Antibodies and affinities can bephenotypically characterized and compared using functional assays (e.g.,flow cytometry assay).

The term “chimeric antigen receptor” or “CAR” as used herein refers toan antigen-binding domain that is fused to an intracellular signalingdomain capable of activating or stimulating an immune cell, and incertain embodiments, the CAR also comprises a transmembrane domain. Incertain embodiments, the CAR's antigen-binding domain is composed of asingle chain variable fragment (scFv) derived from fusing the variableheavy and light regions of a murine or humanized monoclonal antibody.Alternatively, scFvs may be used that are derived from Fab's (instead offrom an antibody, e.g., obtained from Fab libraries). In variousembodiments, the scFv is fused to the transmembrane domain and then tothe intracellular signaling domain. “First generation” CARs includethose that solely provide CD3ζ signals upon antigen binding,“Second-generation” CARs include those that provide both co-stimulation(e.g., CD28 or CD137) and activation (CD3ζ). “Third-generation” CARsinclude those that provide multiple co-stimulation (e.g. CD28 and CD137)and activation (CD3ζ). In various embodiments, the CAR is selected tohave high affinity or avidity for the antigen.

The term “chimeric co-stimulating receptor” or “CCR” refers to achimeric receptor that binds to an antigen and provides co-stimulatorysignals, but does not provide a T-cell activation signal. CCR isdescribed in Krause, et al., J. Exp. Med. (1998); 188(4):619-626, andUS20020018783, the contents of which are incorporated by reference intheir entireties. CCRs mimic co-stimulatory signals, but unlike, CARs,do not provide a T-cell activation signal, e.g., CCRs lack a CD3ζpolypeptide.

The term “immunosuppressive activity” is meant induction of signaltransduction or changes in protein expression in a cell (e.g., anactivated immunoresponsive cell) resulting in a decrease in an immuneresponse. Polypeptides known to suppress or decrease an immune responsevia their binding include CD47, PD-1, CTLA-4, and their correspondingligands, including SIRPa, PD-L1, PD-L2, B7-1, and B7-2. Suchpolypeptides are present in the tumor microenvironment and inhibitimmune responses to neoplastic cells. In various embodiments,inhibiting, blocking, or antagonizing the interaction ofimmunosuppressive polypeptides and/or their ligands enhances the immuneresponse of the immunoresponsive cell.

The term “immunostimulatory activity” is meant induction of signaltransduction or changes in protein expression in a cell (e.g., anactivated immunoresponsive cell) resulting in an increase in an immuneresponse. Immunostimulatory activity may include pro-inflammatoryactivity. Polypeptides known to stimulate or increase an immune responsevia their binding include CD28, OX-40, 4-1BB, and their correspondingligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides arepresent in the tumor microenvironment and activate immune responses toneoplastic cells. In various embodiments, promoting, stimulating, oragonizing pro-inflammatory polypeptides and/or their ligands enhancesthe immune response of the immunoreponsive cell.

By “OX40L polypeptide” is meant a polypeptide having at least about 85%,about 90 about, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to NCBI Reference No: BAB18304 or NP_003317 (SEQID NO: 4) or fragments thereof, and/or may optionally comprise up to oneor up to two or up to three conservative amino acid substitutions. SEQID NO: 4 is provided below

[SEQ ID NO: 4] 1 MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSALQVSHRYPRIQ 61 SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFSQEVNISLHYQ 121 KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGELILIHQNPGEF 181 CVL

By “OX40L nucleic acid molecule” is meant a polynucleotide encoding aOX40L polypeptide.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%homolgous or identical with an endogenous nucleic acid sequence, butwill typically exhibit substantial identity. Polynucleotides having“substantial identity” or “substantial homology” to an endogenoussequence are typically capable of hybridizing with at least one strandof a double-stranded nucleic acid molecule. By “hybridize” is meant pairto form a double-stranded molecule between complementary polynucleotidesequences (e.g., a gene described herein), or portions thereof, undervarious conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In certain embodiments, wash stepswill occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1%SDS. In a more preferred embodiment, wash steps will occur at 68° C. in15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “substantially identical” or “substantially homologous” is meant apolypeptide or nucleic acid molecule exhibiting at least about 50%homolougs or identical to a reference amino acid sequence (for example,any one of the amino acid sequences described herein) or nucleic acidsequence (for example, any one of the nucleic acid sequences describedherein). Preferably, such a sequence is at least about 60%, about 80%,about 85%, about 90%, about 95%, about 99%, or about 100% homolgous oridentical at the amino acid level or nucleic acid to the sequence usedfor comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e-3 and e-100 indicating a closely related sequence.

By “analog” is meant a structurally related polypeptide or nucleic acidmolecule having the function of a reference polypeptide or nucleic acidmolecule.

The term “ligand” as used herein refers to a molecule that binds to areceptor. In particular, the ligand binds a receptor on another cell,allowing for cell-to-cell recognition and/or interaction.

The term “constitutive expression” as used herein refers to expressionunder all physiological conditions.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include neoplasia or pathogen infection of cell.

By “effective amount” is meant an amount sufficient to have atherapeutic effect. In certain embodiments, an “effective amount” is anamount sufficient to arrest, ameliorate, or inhibit the continuedproliferation, growth, or metastasis (e.g., invasion, or migration) ofdisease or disorder of interest, e.g., a myeloid disorder.

By “endogenous” is meant a nucleic acid molecule or polypeptide that isnormally expressed in a cell or tissue.

By “enforcing tolerance” is meant preventing the activity ofself-reactive cells or immunoresponsive cells that target transplantedorgans or tissues.

By “exogenous” is meant a nucleic acid molecule or polypeptide that isnot endogenously present in the cell. The term “exogenous” wouldtherefore encompass any recombinant nucleic acid molecule or polypeptideexpressed in a cell, such as foreign, heterologous, and over-expressednucleic acid molecules and polypeptides. By “exogenous” nucleic acid ismeant a nucleic acid not present in a native wild type cell; for examplean exogenous nucleic acid may vary from an endogenous counterpart bysequence, by position/location, or both. For clarity, an exogenousnucleic acid may have the same or different sequence relative to itsnative endogenous counterpart; it may be introduced by geneticengineering into the cell itself or a progenitor thereof, and mayoptionally be linked to alternative control seqiences, such as anon-native promoter or secretory sequence.

By a “heterologous nucleic acid molecule or polypeptide” is meant anucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptidethat is not normally present in a cell or sample obtained from a cell.This nucleic acid may be from another organism, or it may be, forexample, an mRNA molecule that is not normally expressed in a cell orsample.

By “immunoresponsive cell” is meant a cell that functions in an immuneresponse or a progenitor, or progeny thereof.

By “increase” is meant to alter positively by at least 5%. An alterationmay be by about 5%, about 10%, about 25%, about 30%, about 50%, about75%, about 100% or more.

By “isolated cell” is meant a cell that is separated from the molecularand/or cellular components that naturally accompany the cell.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide of this invention is purified if it is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Purity and homogeneity aretypically determined using analytical chemistry techniques, for example,polyacrylamide gel electrophoresis or high performance liquidchromatography. The term “purified” can denote that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.For a protein that can be subjected to modifications, for example,phosphorylation or glycosylation, different modifications may give riseto different isolated proteins, which can be separately purified.

The term “obtaining” as in “obtaining the agent” is intended to includepurchasing, synthesizing or otherwise acquiring the agent (or indicatedsubstance or material).

“Linker”, as used herein, shall mean a functional group (e.g., chemicalor polypeptide) that covalently attaches two or more polypeptides ornucleic acids so that they are connected to one another. As used herein,a “peptide linker” refers to one or more amino acids used to couple twoproteins together (e.g., to couple V_(H) and V_(L) domains). Anexemplary linker sequence used in the invention is GGGGSGGGGSGGGGS [SEQID NO: 5].

By “modulate” is meant positively or negatively alter. Exemplarymodulations include a about 1%, about 2%, about 5%, about 10%, about25%, about 50%, about 75%, or about 100% change.

By “reduce” is meant to alter negatively by at least about 5%. Analteration may be by about 5%, about 10%, about 25%, about 30%, about50%, about 75%, or even by about 100%.

By “recognize” is meant selectively binds a target. A T cell thatrecognizes aan antigentypically comprises or expresses a receptor thatbinds to that antigen.

By “signal sequence” or “leader sequence” is meant a peptide sequence(e.g., 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus ofnewly synthesized proteins that directs their entry to the secretorypathway. Exemplary leader sequences include, but is not limited to, thekappa leader sequence: METPAQLLFLLLLWLPDTTG [SEQ ID NO:6] (human),METDTLLLWVLLLWVPGSTG [SEQ ID NO:7] (mouse); and the CD8 leader sequence:MALPVTALLLPLALLLHAARP [SEQ ID NO:8] (human).

By “soluble” is meant a polypeptide that is freely diffusible in anaqueous environment (e.g., not membrane bound).

By “specifically binds” is meant a polypeptide or fragment thereof thatrecognizes and binds a biological molecule of interest (e.g., apolypeptide), but which does not substantially recognize and bind othermolecules in a sample, for example, a biological sample, which naturallyincludes a polypeptide of the invention. In certain embodiments,“specifically binds” refers to binding of, for example, an antibody toan epitope or antigen or antigenic determinant in such a manner thatbinding can be displaced or competed with a second preparation ofidentical or similar epitope, antigen or antigenic determinant.

The terms “comprises”, “comprising”, and are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the disease course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Therapeutic effects of treatment include,without limitation, preventing occurrence or recurrence of disease,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, reducing or preventingmetastases, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Bypreventing progression of a disease or disorder, a treatment can reduceor prevent deterioration due to a disorder in an affected or diagnosedsubject or a subject suspected of having the disorder, but also atreatment may prevent the onset of the disorder or a symptom of thedisorder in a subject at risk for the disorder or suspected of havingthe disorder.

The term “subject” as used herein refers to a vertebrate, preferably amammal, more preferably a human. Non-human subjects include non-humanprimates, dogs, cats, horses, rodents, etc.

The term “immunocompromised” as used herein refers to a subject who hasan immunodeficiency. The subject is very vulnerable to opportunisticinfections, infections caused by organisms that usually do not causedisease in a person with a healthy immune system, but can affect peoplewith a poorly functioning or suppressed immune system.

Other aspects of the invention are described in the following disclosureand are within the ambit of the invention.

Antibodies

The present disclosure provides antibodies or antigen-binding portionsthereof that bind to a myeloid/AML antigen.

Antibodies for use in the presently disclosed subject matter include anyantibody, whether natural or synthetic, full length or a fragmentthereof, monoclonal or polyclonal, that binds sufficiently strongly andspecifically to a myeloid/AML antigen. An antibody can have a K_(d) ofat most about 10⁻⁶M, about 10⁻⁷M, about 10⁻⁸M, about 10⁻⁹M, about10⁻¹⁰M, about 10⁻¹¹M and about 10⁻¹²M.

Antibodies and derivatives thereof that can be used encompassespolyclonal or monoclonal antibodies, chimeric, human, humanized,primatized (CDR-grafted), veneered or single-chain antibodies, phaseproduced antibodies (e.g., from phage display libraries), as well asfunctional binding fragments, of antibodies. For example, antibodyfragments capable of binding to a myeloid/AML antigen, or portionsthereof, including, but not limited to Fv, Fab, Fab′ and F(ab′)₂fragments can be used. Such fragments can be produced by enzymaticcleavage or by recombinant techniques. For example, and not by way oflimitation, papain or pepsin cleavage can generate Fab or F(ab′)₂fragments, respectively. Other proteases with the requisite substratespecificity can also be used to generate Fab or F(ab′)₂ fragments.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a chimeric gene encodinga F(ab′)₂ heavy chain portion can be designed to include DNA sequencesencoding the CH, domain and hinge region of the heavy chain.

Methods of raising an antibody targeting a specific antigen aregenerally known in the art. Synthetic and engineered antibodies aredescribed in, e.g., Cabilly et al., U.S. Pat. No. 4,816,567 Cabilly etal., European Patent No. 0125023 B1; Boss et al., U.S. Pat. No.4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M.S. et al., WO 86/01533; Neuberger et al., European Patent No. 0,194,276B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No.0,239,400 B1; Queen et al., European Patent No. 0451216 B1; and Padlanet al., EP 0519596 A1. See also, Newman et al., BioTechnology, 10:1455-1460 (1992), regarding primatized antibody, and Ladner et al., U.S.Pat. No. 4,946,778 and Bird et al., Science, 242: 423-426 (1988))regarding single-chain antibodies. The contents of those publicationsare incorporated by reference in their entireties.

In certain embodiments, one or more of the flowing commerciallyavailable antibodies can be used for binding to a myeloid/AML antigen:CD70-PE cat.355104 (Biolegend); EMR2-FITC cat.130-104-654; EMR2-APC cat.130-104-656 (Milteny); LTB4R1-AF700 cat.FABO99N; LTB4R1-AF405cat.FAB099V; LTB4R1-FITC cat.NB100-64832 (Novus Biologicals); LTB4R1-PEcat. FAB099P (R&D); PIEZO1-AF488 cat.NBP11-78537; CD33-APC cat.551378(BD Pharmingen); ENG-APC cat. MHCD10505 (Invitrogen); MYADMcat.NBP2-24494SS (Novus); ITGA5 (CD49e)-APC cat. 328011 (Biolegend);SLC19A1-APC cat. FAB8450A (R&D); ILT3-APC (LILRB4) cat. FAB24251A (R&D);CCR1-PE cat. 130-100-368 (Milteniy); ITGA4-APC cat. FAB2450A (R&D);CD49d-PE cat. 130-099-691 (Miltenyi); ICAM1-PE cat. 130-103-909(Miltenyi); SIRPB1-PE cat. 130-105-310 (Miltenyi); CD64-APC (FCGR1A)cat. 561189 (BD); CD300f (IREM-1)-PE cat.130-098-472; CD300f(IREM-1)-FITC cat.130-098-443 (Miltenyi); IL10RB-APC cat. FAB874A (R&D);MRP1-PE cat. IC19291P (R&D); CD38− APC cat. MHCD3805; CD38− PE cat.MHCD3804 (Invitrogen); CD34-APC cat. 340667 (BD); CPM cat.DDX0520P(Dendritics); TTYH3 cat. NBP1-91350 (Novus); SLC NHE1 (SLC9A1) ab58304(abcam); SLC22A5 bs-8149R (Bioss); KCNN4 PA5-33875 (Thermo Scientific);ITFG3 PA5-31403 (Thermo Scientific); SLC6A6 LS-C179237 (LSBio); SLC43A3NBP1-85026 (Novus); TFR2 TA504592 (Origene); MBOAT7 NBP1-69610 (Novus);CD235a-APC (GYPA) cat. 551336 (BD Pharmigen); and PLXNC1 cat. AF3887-SP(R&D Systems).

The CDRs of the commercially available antibodies are readily accessibleby one skilled in the art using conventional sequencing technology.Further, one skilled in the art is able to construct nucleic acidsencoding scFvs and antigen recognizing receptors (e.g., CARs and TCRs)based on the CDRs of those antibodies.

T-Cell Receptor (TCR)

The present disclosure provides antigen binding receptors that bind to amyeloid/AML antigen. In certain embodiments, the antigen recognizingreceptor is a TCR. A TCR is a disulfide-linked heterodimeric proteinconsisting of two variable chains expressed as part of a complex withthe invariant CD3 chain molecules. A TCR is found on the surface of Tcells, and is responsible for recognizing antigens as peptides bound tomajor histocompatibility complex (MHC) molecules. In certainembodiments, a TCR comprises an alpha chain and a beta chain (encoded byTRA and TRB, respectively). In certain embodiments, a TCR comprises agamma chain and a delta chain (encoded by TRG and TRD, respectively).

Each chain of a TCR is composed of two extracellular domains: Variable(V) region and a Constant (C) region. The Constant region is proximal tothe cell membrane, followed by a transmembrane region and a shortcytoplasmic tail. The Variable region binds to the peptide/MHC complex.The variable domain of both chains each has three complementaritydetermining regions (CDRs).

In certain embodiments, a TCR can form a receptor complex with threedimeric signaling modules CD3δ/ε, CD3γ/ε and CD247ζ/ζ or ζ/η. When a TCRcomplex engages with its antigen and MHC (peptide/MHC), the T cellexpressing the TCR complex is activated.

In certain embodiments, the presently disclosed subject matter providesa recombinant TCR. In certain embodiments, the TCR is a non-naturallyoccurring TCR. In certain embodiments, the TCR differs from anynaturally occurring TCR by at least one amino acid residue. In certainembodiments, the TCR differs from any naturally occurring TCR by atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40,50, 60, 70, 80, 90, 100 or more amino acid residues. In certainembodiments, the TCR is modified from a naturally occurring TCR by atleast one amino acid residue. In certain embodiments, the TCR ismodified from a naturally occurring TCR by at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 ormore amino acid residues.

Chimeric Antigen Receptor (CAR)

The present disclosure further provides chimeric antigen receptors(CARs) that target a myeloid/AML antigen.

CARs are engineered receptors, which graft or confer a specificity ofinterest onto an immune effector cell. CARs can be used to graft thespecificity of a monoclonal antibody onto a T cell; with transfer oftheir coding sequence facilitated by retroviral vectors.

There are three generations of CARs. “First generation” CARs aretypically composed of an extracellular antigen binding domain (e.g., asingle-chain variable fragments (scFv)) fused to a transmembrane domain,fused to cytoplasmic/intracellular signaling domain of the T cellreceptor chain. “First generation” CARs typically have the intracellularsignaling domain from the CD3ζ-chain, which is the primary transmitterof signals from endogenous TCRs. “First generation” CARs can provide denovo antigen recognition and cause activation of both CD4⁺ and CD8⁺ Tcells through their CD3ζ chain signaling domain in a single fusionmolecule, independent of HLA-mediated antigen presentation. “Secondgeneration” CARs add intracellular signaling domains from variousco-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to thecytoplasmic tail of the CAR to provide additional signals to the T cell.“Second generation” CARs comprise those that provide both co-stimulation(e.g., CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies haveindicated that “Second Generation” CARs can improve the anti-tumoractivity of T cells. For example, robust efficacy of “Second Generation”CAR modified T cells was demonstrated in clinical trials targeting theCD19 molecule in patients with chronic lymphoblastic leukemia (CLL) andacute lymphoblastic leukemia (ALL). “Third generation” CARs comprisethose that provide multiple co-stimulation (e.g., CD28 and 4-1BB) andactivation (CD3ζ).

In certain non-limiting embodiments, the extracellular antigen-bindingdomain of the CAR (embodied, for example, an scFv or an analog thereof)binds to an AML antigen with a dissociation constant (K_(d)) of about2×10⁻⁷ M or less. In certain embodiments, the K_(d) is about 2×10⁻⁷ M orless, about 1×10⁻⁷ M or less, about 9×10⁻⁸ M or less, about 1×10⁻⁸ M orless, about 9×10⁻⁹ M or less, about 5×10⁻⁹ M or less, about 4×10⁻⁹ M orless, about 3×10⁻⁹ or less, about 2×10⁻⁹ M or less, or about 1×10⁻⁹ M orless. In certain non-limiting embodiments, the K_(d) is about 3×10⁻⁹ Mor less. In certain non-limiting embodiments, the K_(d) is from about1×10⁻⁹ M to about 3×10⁻⁷ M. In certain non-limiting embodiments, theK_(d) is from about 1.5×10⁻⁹ M to about 3×10⁻⁷ M. In certainnon-limiting embodiments, the K_(d) is from about 1.5×10⁻⁹ M to about2.7×10⁻⁷ M.

Binding of the extracellular antigen-binding domain (for example, in anscFv or an analog thereof) of a presently disclosed AML-targeted CAR canbe confirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetect the presence of protein-antibody complexes of particular interestby employing a labeled reagent (e.g., an antibody, or a scFv) specificfor the complex of interest. For example, the scFv can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography. In certain embodiments, the extracellularantigen-binding domain of the AML antigen-targeted CAR is labeled with afluorescent marker. Non-limiting examples of fluorescent markers includegreen fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP,EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP,Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP,Citrine, Venus, and YPet). In one embodiment, the scFv of a presentlydisclosed AML antigen-targeted CAR is labeled with GFP.

In accordance with the presently disclosed subject matter, the CARscomprise an extracellular antigen-binding domain, a transmembrane domainand an intracellular signaling domain, wherein the extracellularantigen-binding domain specifically binds to an AML antigen. In certainembodiments, the extracellular antigen-binding domain is an scFv. Incertain embodiments, the extracellular antigen-binding domain is a Fab,which is optionally crosslinked. In a certain embodiments, theextracellular binding domain is a F(ab)₂. In certain embodiments, any ofthe foregoing molecules may be comprised in a fusion protein with aheterologous sequence to form the extracellular antigen-binding domain.In certain embodiments, the scFv is identified by screening scFv phagelibrary with an AML antigen-Fc fusion protein.

Extracellular Antigen-Binding Domain of a CAR

In certain embodiments, the extracellular antigen-binding domainspecifically binds to an AML antigen. In certain embodiments, the AMLantigen is a human polypeptide. In certain embodiments, theextracellular antigen-binding domain is an scFv. In certain embodiments,the scFv is a human scFv. In certain embodimens, the scFv is a humanizedscFv.

Transmembrane Domain of a CAR

In certain non-limiting embodiments, the transmembrane domain of the CARcomprises a hydrophobic alpha helix that spans at least a portion of themembrane. Different transmembrane domains result in different receptorstability. After antigen recognition, receptors cluster and a signal istransmitted to the cell. In accordance with the presently disclosedsubject matter, the transmembrane domain of the CAR can comprise a CD8polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide,a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide,a BTLA polypeptide, a synthetic peptide (not based on a proteinassociated with the immune response), or a combination thereof.

In certain embodiments, the transmembrane domain comprises a CD8polypeptide. In certain embodiments, the CD8 polypeptide has an aminoacid sequence that is at least about 85%, about 90%, about 95%, about96%, about 97%, about 98%, about 99% or about 100% homologous to thesequence having a NCBI Reference No: NP_001139345.1 (SEQ ID NO: 9)(homology herein may be determined using standard software such as BLASTor FASTA) as provided below, or fragments thereof, and/or may optionallycomprise up to one or up to two or up to three conservative amino acidsubstitutions. In certain embodiments, the CD8 polypeptide can have anamino acid sequence that is a consecutive portion of SEQ ID NO: 9 whichis at least 20, or at least 30, or at least 40, or at least 50, and upto 235 amino acids in length. Alternatively or additionally, innon-limiting various embodiments, the CD8 polypeptide comprises or hasan amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100to 150, 150 to 200, or 200 to 235 of SEQ ID NO: 9. In certainembodiments, the CAR of the presently disclosed comprises atransmembrane domain comprising a CD8 polypeptide that comprises anamino acid sequence of amino acids 137 to 209 of SEQ ID NO: 9.

[SEQ ID NO: 9] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

In certain embodiments, the CD8 polypeptide has an amino acid sequencethat is at least about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99% or about 100% homologous to the sequence having aNCBI Reference No: AAA92533.1 (SEQ ID NO: 10) (homology herein may bedetermined using standard software such as BLAST or FASTA) as providedbelow, or fragments thereof, and/or may optionally comprise up to one orup to two or up to three conservative amino acid substitutions. Incertain embodiments, the CD8 polypeptide can have an amino acid sequencethat is a consecutive portion of SEQ ID NO: 10 which is at least about20, or at least about 30, or at least about 40, or at least about 50, orat least about 60, or at least about 70, or at least about 100, or atleast about 200, and up to 247 amino acids in length. Alternatively oradditionally, in non-limiting various embodiments, the CD8 polypeptidecomprises or has an amino acid sequence of amino acids 1 to 247, 1 to50, 50 to 100, 100 to 150, 150 to 200, 151 to 219, or 200 to 247 of SEQID NO: 10. In certain embodiments, the CAR of the presently disclosedcomprises a transmembrane domain comprising a CD8 polypeptide thatcomprises an amino acid sequence of amino acids 151 to 219 of SEQ ID NO:10.

[SEQ ID NO: 10] 1 MASPLTRFLS LNLLLMGESI ILGSGEAKPQ APELRIFPKK MDAELGQKVDLVCEVLGSVS 61 QGCSWLFQNS SSKLPQPTFV VYMASSHNKI TWDEKLNSSK LFSAVRDTNNKYVLTLNKFS 121 KENEGYYFCS VISNSVMYFS SVVPVLQKVN STTTKPVLRT PSPVHPTGTSQPQRPEDCRP 181 RGSVKGTGLD FACDIYIWAP LAGICVAPLL SLIITLICYH RSRKRVCKCPRPLVRQEGKP 241 RPSEKIV

In certain embodiments, the CD8 polypeptide comprises or has the aminoacid sequence set forth in SEQ ID NO: 11, which is provided below:

[SEQ ID NO: 11] STTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICY

In accordance with the presently disclosed subject matter, a “CD8nucleic acid molecule” refers to a polynucleotide encoding a CD8polypeptide.

In certain embodiments, the CD8 nucleic acid molecule encoding the CD8polypeptide comprised in the transmembrane domain of the presentlydisclosed CAR (SEQ ID NO: 11) comprises nucleic acids having thesequence set forth in SEQ ID NO: 12 as provided below.

[SEQ ID NO: 12] TCTACTACTACCAAGCCAGTGCTGCGAACTCCCTCACCTGTGCACCCTACCGGGACATCTCAGCCCCAGAGACCAGAAGATTGTCGGCCCCGTGGCTCAGTGAAGGGGACCGGATTGGACTTCGCCTGTGATATTTACATCTGGGCACCCTTGGCCGGAATCTGCGTGGCCCTTCTGCTGTCCTTGATCATCACTCTCAT CTGCTAC

In certain embodiments, the transmembrane domain of a presentlydisclosed CAR comprises a CD28 polypeptide. The CD28 polypeptide canhave an amino acid sequence that is at least about 85%, about 90%, about95%, about 96%, about 97%, about 98%, about 99% or 100% homologous tothe sequence having a NCBI Reference No: P10747 or NP_006130 (SEQ ID No:2), or fragments thereof, and/or may optionally comprise up to one or upto two or up to three conservative amino acid substitutions. Innon-limiting certain embodiments, the CD28 polypeptide can have an aminoacid sequence that is a consecutive portion of SEQ ID NO: 2 which is atleast 20, or at least 30, or at least 40, or at least 50, and up to 220amino acids in length. Alternatively or additionally, in non-limitingvarious embodiments, the CD28 polypeptide has an amino acid sequence ofamino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to200, or 200 to 220 of SEQ ID NO: 2. In certain embodiments, the CD28polypeptide comprised in the transmembrane domain of a presentlydisclosed CAR has an amino acid sequence of amino acids 153 to 179 ofSEQ ID NO: 2.

SEQ ID NO: 2 is provided below:

[SEQ ID NO: 2] 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSREFRASLHKGLD 61 SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIYFCKIEVMYPP 121 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLVTVAFIIFWVR 181 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS

In accordance with the presently disclosed subject matter, a “CD28nucleic acid molecule” refers to a polynucleotide encoding a CD28polypeptide.

In certain non-limiting embodiments, a CAR can also comprise a spacerregion that links the extracellular antigen-binding domain to thetransmembrane domain. The spacer region can be flexible enough to allowthe antigen binding domain to orient in different directions tofacilitate antigen recognition. The spacer region can be the hingeregion from IgG1, or the CH₂CH₃ region of immunoglobulin and portions ofCD3.

Intracellular Signaling Domain of a CAR

In certain non-limiting embodiments, an intracellular signaling domainof the CAR can comprise a CD3ζ polypeptide, which can activate orstimulate a cell (e.g., a cell of the lymphoid lineage, e.g., a T cell).CD3ζ comprises 3 ITAMs, and transmits an activation signal to the cell(e.g., a cell of the lymphoid lineage, e.g., a T cell) after antigen isbound. In certain embodiments, the CD3ζ polypeptide has an amino acidsequence that is at least about 85%, about 90%, about 95%, about 96%,about 97%, about 98%, about 99% or about 100% homologous to the sequencehaving a NCBI Reference No: NP_932170 (SEQ ID No: 1), or fragmentsthereof, and/or may optionally comprise up to one or up to two or up tothree conservative amino acid substitutions. In certain non-limitingembodiments, the CD3ζ polypeptide can have an amino acid sequence thatis a consecutive portion of SEQ ID NO: 1 which is at least 20, or atleast 30, or at least 40, or at least 50, and up to 164 amino acids inlength. Alternatively or additionally, in non-limiting variousembodiments, the CD3ζ polypeptide has an amino acid sequence of aminoacids 1 to 164, 1 to 50, 50 to 100, 100 to 150, or 150 to 164 of SEQ IDNO: 1. In certain embodiments, the CD3ζ polypeptide comprises or has anamino acid sequence of amino acids 52 to 164 of SEQ ID NO: 1.

SEQ ID NO: 1 is provided below:

[SEQ ID NO: 1] 1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRVKFSRSAD 61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGLYNELQKDKMA 121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR

In certain embodiments, the CD3ζ polypeptide has an amino acid sequencethat is at least about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99% or about 100% homologous to the sequence having aNCBI Reference No: NP_001106864.2 (SEQ ID No: 13), or fragments thereof,and/or may optionally comprise up to one or up to two or up to threeconservative amino acid substitutions. In certain non-limitingembodiments, the CD3ζ polypeptide can have an amino acid sequence thatis a consecutive portion of SEQ ID NO: 13 which is at least about 20, orat least about 30, or at least about 40, or at least about 50, or atleast about 90, or at least about 100, and up to 188 amino acids inlength. Alternatively or additionally, in non-limiting variousembodiments, the CD3ζ polypeptide has an amino acid sequence of aminoacids 1 to 164, 1 to 50, 50 to 100, 52 to 142, 100 to 150, or 150 to 188of SEQ ID NO: 13. In certain embodiments, the CD3ζ polypeptide comprisesor has an amino acid sequence of amino acids 52 to 142 of SEQ ID NO: 13.

SEQ ID NO: 13 is provided below:

[SEQ ID NO: 13] 1 MKWKVSVLAC ILHVRFPGAE AQSFGLLDPK LCYLLDGILF IYGVIITALYLRAKFSRSAE 61 TAANLQDPNQ LYNELNLGRR EEYDVLEKKR ARDPEMGGKQ RRRNPQEGVYNALQKDKMAE 121 AYSEIGTKGE RRRGKGHDGL YQDSHFQAVQ FGNRREREGS ELTRTLGLRARPKACRHKKP 181 LSLPAAVS

In certain embodiments, the CD3ζ polypeptide comprises or has the aminoacid sequence set forth in SEQ ID NO: 14, which is provided below:

[SEQ ID NO: 14] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKD TYDALHMQTLAPR

In accordance with the presently disclosed subject matter, a “CD3ζnucleic acid molecule” refers to a polynucleotide encoding a CD3ζpolypeptide. In certain embodiments, the CD3ζ nucleic acid moleculeencoding the CD3ζ polypeptide comprised in the intracellular domain of apresently disclosed CAR (SEQ ID NO: 14) comprises the nucleotidesequence set forth in SEQ ID NO: 15 as provided below.

[SEQ ID NO: 15] AGAGCAAAATTCAGCAGGAGTGCAGAGACTGCTGCCAACCTGCAGGACCCCAACCAGCTCTACAATGAGCTCAATCTAGGGCGAAGAGAGGAATATGACGTCTTGGAGAAGAAGCGGGCTCGGGATCCAGAGATGGGAGGCAAACAGCAGAGGAGGAGGAACCCCCAGGAAGGCGTATACAATGCACTGCAGAAAGACAAGATGGCAGAAGCCTACAGTGAGATCGGCACAAAAGGCGAGAGGCGGAGAGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGCACTGCCACCAAGGACACCTATGATGCCCTGCATATGCAGACCCTGGCCCCTCGCTAA

In certain non-limiting embodiments, an intracellular signaling domainof the CAR further comprises at least one signaling region. The at leastone signaling region can include a CD28 polypeptide, a 4-1BBpolypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide(not based on a protein associated with the immune response), or acombination thereof.

In certain embodiments, the signaling region is a co-stimulatorysignaling region. In certain embodiments, the co-stimulatory regioncomprises at least one co-stimulatory molecule, which can provideoptimal lymphocyte activation. As used herein, “co-stimulatorymolecules” refer to cell surface molecules other than antigen receptorsor their ligands that are required for an efficient response oflymphocytes to antigen. The at least one co-stimulatory signaling regioncan include a CD28 polypeptide, a 4-1BB polypeptide, an OX40polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combinationthereof. The co-stimulatory molecule can bind to a co-stimulatoryligand, which is a protein expressed on cell surface that upon bindingto its receptor produces a co-stimulatory response, i.e., anintracellular response that effects the stimulation provided when anantigen binds to its CAR molecule. Co-stimulatory ligands, include, butare not limited to CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, andPD-L1. As one example, a 4-1BB ligand (i.e., 4-1BBL) may bind to 4-1BB(also known as “CD137”) for providing an intracellular signal that incombination with a CAR signal induces an effector cell function of theCAR⁺ T cell. CARs comprising an intracellular domain that comprises aco-stimulatory signaling region comprising 4-1BB, ICOS or DAP-10 aredisclosed in U.S. Pat. No. 7,446,190 (e.g., the nucleotide sequenceencoding 4-1BB is set forth in SEQ ID NO:15, the nucleotide sequenceencoding ICOS is set forth in SEQ ID NO:16, and the nucleotide sequenceencoding DAP-10 is set forth in SEQ ID NO:17 in U.S. Pat. No.7,446,190), which is herein incorporated by reference in its entirety.

In certain embodiments, the intracellular signaling domain of the CARcomprises a co-stimulatory signaling region that comprises a CD28polypeptide. The CD28 polypeptide can have an amino acid sequence thatis at least about 85%, about 90%, about 95%, about 96%, about 97%, about98%, about 99% or 100% homologous to the sequence having a NCBIReference No: P10747 or NP_006130 (SEQ ID No: 2), or fragments thereof,and/or may optionally comprise up to one or up to two or up to threeconservative amino acid substitutions. In non-limiting certainembodiments, the CD28 polypeptide has an amino acid sequence that is aconsecutive portion of SEQ ID NO: 2 which is at least 20, or at least30, or at least 40, or at least 50, and up to 220 amino acids in length.Alternatively or additionally, in non-limiting various embodiments, theCD28 polypeptide has an amino acid sequence of amino acids 1 to 220, 1to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 ofSEQ ID NO: 2. In certain embodiments, the intracellular signaling domainof the CAR comprises a co-stimulatory signaling region that comprises aCD28 polypeptide having an amino acid sequence of amino acids 180 to 220of SEQ ID NO: 2.

In certain embodiments, the CD28 polypeptide has an amino acid sequencethat is at least about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99% or about 100% homologous to the sequence having aNCBI Reference No: NP_031668.3 (SEQ ID No: 16), or fragments thereof,and/or may optionally comprise up to one or up to two or up to threeconservative amino acid substitutions. In non-limiting certainembodiments, the CD28 polypeptide has an amino acid sequence that is aconsecutive portion of SEQ ID NO: 16 which is at least about 20, or atleast about 30, or at least about 40, or at least about 50, and up to218 amino acids in length. Alternatively or additionally, innon-limiting various embodiments, the CD28 polypeptide has an amino acidsequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 114 to220, 150 to 200, 178 to 218, or 200 to 220 of SEQ ID NO: 16. In certainembodiments, the co-stimulatory signaling region of a presentlydisclosed CAR comprises a CD28 polypeptide that comprises or has theamino acids 178 to 218 of SEQ ID NO: 16.

SEQ ID NO: 16 is provided below:

[SEQ ID NO: 16] 1 MTLRLLFLAL NFFSVQVTEN KILVKQSPLL VVDSNEVSLS CRYSYNLLAKEFRASLYKGV 61 NSDVEVCVGN GNFTYQPQFR SNAEFNCDGD FDNETVTFRL WNLHVNHTDIYFCKIEFMYP 121 PPYLDNERSN GTIIHIKEKH LCHTQSSPKL FWALVVVAGV LFCYGLLVTVALCVIWTNSR 181 RNRLLQSDYM NMTPRRPGLT RKPYQPYAPA RDFAAYRP

In accordance with the presently disclosed subject matter, a “CD28nucleic acid molecule” refers to a polynucleotide encoding a CD28polypeptide. In certain embodiments, a CD28 nucleic acid molecule thatencodes a CD28 polypeptide comprised in the co-stimulatory signalingregion of a presently disclosed CAR (e.g., amino acids 178 to 218 of SEQID NO: 16) comprises or has a nucleotide sequence set forth in SEQ IDNO: 17, which is provided below.

[SEQ ID NO: 17] AATAGTAGAAGGAACAGACTCCTTCAAAGTGACTACATGAACATGACTCCCCGGAGGCCTGGGCTCACTCGAAAGCCTTACCAGCCCTACGCCCCTGCCAGAGACTTTGCAGCGTACCGCCCC

In certain embodiments, the intracellular domain of the CAR comprises aco-stimulatory signaling region that comprises two co-stimulatorymolecules: CD28 and 4-1BB or CD28 and OX40.

4-1BB can act as a tumor necrosis factor (TNF) ligand and havestimulatory activity. The 4-1BB polypeptide can have an amino acidsequence that is at least about 85%, about 90%, about 95%, about 96%,about 97%, about 98%, about 99% or about 100% homologous to the sequencehaving a NCBI Reference No: P41273 or NP_001552 (SEQ ID NO: 3) orfragments thereof, and/or may optionally comprise up to one or up to twoor up to three conservative amino acid substitutions.

SEQ ID NO: 3 is provided below:

[SEQ ID NO: 3] 1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPPNSFSSAGGQR 61 TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQELTKKGCKDC 121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGASSVTPPAPARE 181 PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMRPVQTTQEEDG 241 CSCRFPEEEE GGCEL

In accordance with the presently disclosed subject matter, a “4-1BBnucleic acid molecule” refers to a polynucleotide encoding a 4-1BBpolypeptide.

An OX40 polypeptide can have an amino acid sequence that is at leastabout 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99% or about 100% homologous to the sequence having a NCBI Reference No:P43489 or NP_003318 (SEQ ID NO: 18), or fragments thereof, and/or mayoptionally comprise up to one or up to two or up to three conservativeamino acid substitutions.

SEQ ID NO: 18 is provided below:

[SEQ ID NO: 18] 1 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGNGMVSRCSRSQ 61 NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCRAGTQPLDSYK 121 PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRDPPATQPQETQ 181 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGPLAILLALYLL 241 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI

In accordance with the presently disclosed subject matter, an “OX40nucleic acid molecule” refers to a polynucleotide encoding an OX40polypeptide.

An ICOS polypeptide can have an amino acid sequence that is at leastabout 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99% or about 100% homologous to the sequence having a NCBI Reference No:NP_036224 (SEQ ID NO: 19) or fragments thereof, and/or may optionallycomprise up to one or up to two or up to three conservative amino acidsubstitutions.

SEQ ID NO: 19 is provided below:

[SEQ ID NO: 19] 1 MKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQFKMQLLKGGQ 61 ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLD HSHANYYFCNLSIFDPPPFK 121 VTLTGGYLHI YESQLCCQLK FWLPIGCAAF VVVCILGCIL ICWLTKKKYSSSVHDPNGEY 181 MFMRAVNTAK KSRLTDVTL

In accordance with the presently disclosed subject matter, an “ICOSnucleic acid molecule” refers to a polynucleotide encoding an ICOSpolypeptide.

CTLA-4 is an inhibitory receptor expressed by activated T cells, whichwhen engaged by its corresponding ligands (CD80 and CD86; B7-1 and B7-2,respectively), mediates activated T cell inhibition or anergy. In bothpreclinical and clinical studies, CTLA-4 blockade by systemic antibodyinfusion, enhanced the endogenous anti-tumor response albeit, in theclinical setting, with significant unforeseen toxicities.

CTLA-4 contains an extracellular V domain, a transmembrane domain, and acytoplasmic tail. Alternate splice variants, encoding differentisoforms, have been characterized. The membrane-bound isoform functionsas a homodimer interconnected by a disulfide bond, while the solubleisoform functions as a monomer. The intracellular domain is similar tothat of CD28, in that it has no intrinsic catalytic activity andcontains one YVKM motif (SEQ ID NO: 25) able to bind PI3K, PP2A andSHP-2 and one proline-rich motif able to bind SH3 containing proteins.One role of CTLA-4 in inhibiting T cell responses seem to be directlyvia SHP-2 and PP2A dephosphorylation of TCR-proximal signaling proteinssuch as CD3 and LAT. CTLA-4 can also affect signaling indirectly viacompeting with CD28 for CD80/86 binding. CTLA-4 has also been shown tobind and/or interact with PI3K, CD80, AP2M1, and PPP2R5A.

In accordance with the presently disclosed subject matter, a CTLA-4polypeptide can have an amino acid sequence that is at least about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to UniProtKB/Swiss-Prot Ref. No.: P16410.3 (SEQ IDNO: 20) (homology herein may be determined using standard software suchas BLAST or FASTA) or fragments thereof, and/or may optionally compriseup to one or up to two or up to three conservative amino acidsubstitutions.

SEQ ID NO: 20 is provided below:

[SEQ ID NO: 20] 1 MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQPAVVLASSRGIASFVCEY 61 ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGNQVNLTIQGLR 121 AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAVSSGLFFYSFL 181 LTAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN

In accordance with the presently disclosed subject matter, a “CTLA-4nucleic acid molecule” refers to a polynucleotide encoding a CTLA-4polypeptide.

PD-1 is a negative immune regulator of activated T cells upon engagementwith its corresponding ligands PD-L1 and PD-L2 expressed on endogenousmacrophages and dendritic cells. PD-1 is a type I membrane protein of268 amino acids. PD-1 has two ligands, PD-L1 and PD-L2, which aremembers of the B7 family. The protein's structure comprises anextracellular IgV domain followed by a transmembrane region and anintracellular tail. The intracellular tail contains two phosphorylationsites located in an immunoreceptor tyrosine-based inhibitory motif andan immunoreceptor tyrosine-based switch motif, that PD-1 negativelyregulates TCR signals. SHP-I and SHP-2 phosphatases bind to thecytoplasmic tail of PD-1 upon ligand binding. Upregulation of PD-L1 isone mechanism tumor cells may evade the host immune system. Inpre-clinical and clinical trials, PD-1 blockade by antagonisticantibodies induced anti-tumor responses mediated through the hostendogenous immune system.

In accordance with the presently disclosed subject matter, a PD-1polypeptide can have an amino acid sequence that is at least about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to NCBI Reference No: NP_005009.2 (SEQ ID NO: 21)or fragments thereof, and/or may optionally comprise up to one or up totwo or up to three conservative amino acid substitutions.

SEQ ID NO: 21 is provided below:

[SEQ ID NO: 21] 1 MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNATFTCSFSNTS 61 ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSVVRARRNDSGT 121 YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLVVGVVGGLLGS 181 LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQWREKTPEPPVP 241 CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL

In accordance with the presently disclosed subject matter, a “PD-1nucleic acid molecule” refers to a polynucleotide encoding a PD-1polypeptide.

Lymphocyte-activation protein 3 (LAG-3) is a negative immune regulatorof immune cells. LAG-3 belongs to the immunoglobulin (Ig) superfamilyand contains 4 extracellular Ig-like domains. The LAG3 gene contains 8exons. The sequence data, exon/intron organization, and chromosomallocalization all indicate a close relationship of LAG3 to CD4. LAG3 hasalso been designated CD223 (cluster of differentiation 223).

In accordance with the presently disclosed subject matter, a LAG-3polypeptide can have an amino acid sequence that is at least about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to UniProtKB/Swiss-Prot Ref. No.: P18627.5 (SEQ IDNO: 22) or fragments thereof, and/or may optionally comprise up to oneor up to two or up to three conservative amino acid substitutions.

SEQ ID NO: 22 is provided below:

[SEQ ID NO: 22] 1 MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPA QLPCSPTIPLQDLSLLRRAG 61 VTWQHQPDSG PPAAAPGHPL APGPHPAAPS SWGPRPRRYT VLSVGPGGLRSGRLPLQPRV 121 QLDERGRQRG DFSLWLRPAR RADAGEYRAA VHLRDRALSC RLRLRLGQASMTASPPGSLR 181 ASDWVILNCS FSRPDRPASV HWFRNRGQGR VPVRESPHHH LAESFLFLPQVSPMDSGPWG 241 CILTYRDGFN VSIMYNLTVL GLEPPTPLTV YAGAGSRVGL PCRLPAGVGTRSFLTAKWTP 301 PGGGPDLLVT GDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAIITVTPKSFGS 361 PGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPWLEA QEAQLLSQPWQCQLYQGERL 421 LGAAVYFTEL SSPGAQRSGR APGALPAGHL LLFLILGVLS LLLLVTGAFGFHLWRRQWRP 481 RRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL

In accordance with the presently disclosed subject matter, a “LAG-3nucleic acid molecule” refers to a polynucleotide encoding a LAG-3polypeptide.

Natural Killer Cell Receptor 2B4 (2B4) mediates non-MHC restricted cellkilling on NK cells and subsets of T cells. To date, the function of 2B4is still under investigation, with the 2B4-S isoform believed to be anactivating receptor, and the 2B4-L isoform believed to be a negativeimmune regulator of immune cells. 2B4 becomes engaged upon binding itshigh-affinity ligand, CD48. 2B4 contains a tyrosine-based switch motif,a molecular switch that allows the protein to associate with variousphosphatases. 2B4 has also been designated CD244 (cluster ofdifferentiation 244).

In accordance with the presently disclosed subject matter, a 2B4polypeptide can have an amino acid sequence that is at least about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to UniProtKB/Swiss-Prot Ref. No.: Q9BZW8.2 (SEQ IDNO: 23) or fragments thereof, and/or may optionally comprise up to oneor up to two or up to three conservative amino acid substitutions.

SEQ ID NO: 23 is provided below:

[SEQ ID NO: 23] 1 MLGQVVTLIL LLLLKVYQGK GCQGSADHVV SISGVPLQLQ PNSIQTKVDSIAWKKLLPSQ 61 NGFHHILKWE NGSLPSNTSN DRFSFIVKNL SLLIKAAQQQ DSGLYCLEVTSISGKVQTAT 121 FQVFVFESLL PDKVEKPRLQ GQGKILDRGR CQVALSCLVS RDGNVSYAWYRGSKLIQTAG 181 NLTYLDEEVD INGTHTYTCN VSNPVSWESH TLNLTQDCQN AHQEFRFWPFLVIIVILSAL 241 FLGTLACFCV WRRKRKEKQS ETSPKEFLTI YEDVKDLKTR RNHEQEQTFPGGGSTIYSMI 301 QSQSSAPTSQ EPAYTLYSLI QPSRKSGSRK RNHSPSFNST IYEVIGKSQPKAQNPARLSR 361 KELENFDVYS

In accordance with the presently disclosed subject matter, a “2B4nucleic acid molecule” refers to a polynucleotide encoding a 2B4polypeptide.

B- and T-lymphocyte attenuator (BTLA) expression is induced duringactivation of T cells, and BTLA remains expressed on Th1 cells but notTh2 cells. Like PD1 and CTLA4, BTLA interacts with a B7 homolog, B7H4.However, unlike PD-1 and CTLA-4, BTLA displays T-Cell inhibition viainteraction with tumor necrosis family receptors (TNF-R), not just theB7 family of cell surface receptors. BTLA is a ligand for tumor necrosisfactor (receptor) superfamily, member 14 (TNFRSF14), also known asherpes virus entry mediator (HVEM). BTLA-HVEM complexes negativelyregulate T-cell immune responses. BTLA activation has been shown toinhibit the function of human CD8⁺ cancer-specific T cells. BTLA hasalso been designated as CD272 (cluster of differentiation 272).

In accordance with the presently disclosed subject matter, a BTLApolypeptide can have an amino acid sequence that is at least about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to UniProtKB/Swiss-Prot Ref No.: Q7Z6A9.3 (SEQ IDNO: 24) or fragments thereof, and/or may optionally comprise up to oneor up to two or up to three conservative amino acid substitutions.

SEQ ID NO: 24 is provided below:

[SEQ ID NO: 24] 1 MKTLPAMLGT GKLFWVFFLI PYLDIWNIHG KESCDVQLYI KRQSEHSILAGDPFELECPV 61 KYCANRPHVT WCKLNGTTCV KLEDRQTSWK EEKNISFFIL HFEPVLPNDNGSYRCSANFQ 121 SNLIESHSTT LYVTDVKSAS ERPSKDEMAS RPWLLYRLLP LGGLPLLITTCFCLFCCLRR 181 HQGKQNELSD TAGREINLVD AHLKSEQTEA STRQNSQVLL SETGIYDNDPDLCFRMQEGS 241 EVYSNPCLEE NKPGIVYASL NHSVIGPNSR LARNVKEAPT EYASICVRS

In accordance with the presently disclosed subject matter, a “BTLAnucleic acid molecule” refers to a polynucleotide encoding a BTLApolypeptide.

In certain embodiments, the CAR of the presently disclosed subjectmatter can further comprise an inducible promoter, for expressingnucleic acid sequences in human cells. Promoters for use in expressingCAR genes can be a constitutive promoter, such as ubiquitin C (UbiC)promoter.

The presently disclosed subject matter also provides isolated nucleicacid molecule encoding an AML antigen-targeted CAR described herein or afunctional portion thereof. In certain embodiments, the isolated nucleicacid molecule encodes a presently disclosed an AML antigen-targeted CARcomprising an scFv that specifically binds to an AML antigen, atransmembrane domain comprising a CD8 polypeptide, and an intracellulardomain comprising a co-stimulatory signaling region comprising a CD28polypeptide and a CD3ζ polypeptide.

In certain embodiments, the isolated nucleic acid molecule encodes afunctional portion of a presently disclosed an AML antigen-targeted CAR.As used herein, the term “functional portion” refers to any portion,part or fragment of a presently disclosed an AML antigen-targeted CAR,which portion, part or fragment retains the biological activity of anAML antigen-targeted CAR (the parent CAR). For example, functionalportions encompass the portions, parts or fragments of a presentlydisclosed an AML antigen-targeted CAR that retains the ability torecognize a target cell, to treat a disease, e.g., myeloid disorder, toa similar, same, or even a higher extent as the parent CAR. In certainembodiments, an isolated nucleic acid molecule encoding a functionalportion of a presently disclosed an AML antigen-targeted CAR can encodea protein comprising, e.g., about 10%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about95%, or more of the parent CAR.

Chimeric Co-Stimulatory Receptor (CCR)

As used herein, the term “chimeric co-stimulatory receptor” or “CCR”refers to a chimeric receptor that binds to an antigen and providesco-stimulatory signals, but does not provide a T-cell activation signal.CCR is described in Krause, et al., J. Exp. Med. (1998); 188(4):619-626,and US20020018783, the contents of which are incorporated by referencein their entireties. CCRs mimic co-stimulatory signals, but unlike,CARs, do not provide a T-cell activation signal, e.g., CCRs lack a CD3ζpolypeptide. CCRs provide co-stimulation, e.g., a CD28-like signal, inthe absence of the natural co-timulatory ligand on theantigen-presenting cell. A combinatorial antigen recognition, i.e., useof a CCR in combination with a CAR, can augment T-cell reactivityagainst the dual-antigen expressing T cells, thereby improving selectivetumor targeting. Kloss et al., describe a strategy that integratescombinatorial antigen recognition, split signaling, and, critically,balanced strength of T-cell activation and costimulation to generate Tcells that eliminate target cells that express a combination of antigenswhile sparing cells that express each antigen individually (Kloss etal., Nature Biotechnololgy (2013); 31(1):71-75, the content of which isincorporated by reference in its entirety). With this approach, T-cellactivation requires CAR-mediated recognition of one antigen, whereascostimulation is independently mediated by a CCR specific for a secondantigen. To achieve tumor selectivity, the combinatorial antigenrecognition approach diminishes the efficiency of T-cell activation to alevel where it is ineffective without rescue provided by simultaneousCCR recognition of the second antigen.

In certain embodiments, the CCR comprises an extracellularantigen-binding domain that binds to a second antigen, a transmembranedomain, and a co-stimulatory signaling region that comprises at leastone co-stimulatory molecule. In certain embodiments, the CCR does notalone deliver an activation signal tto the cell. Non-limiting examplesof co-stimulatory molecules include CD28, 4-1BB, OX40ICOS, and DAP-10.In certain embodiments, the co-stimulatory signaling region of the CCRcomprises one co-stimulatory signaling molecule. In certainembodiments,the one co-stimulatory signaling molecule is CD28. In certainembodiments, the one co-stimulatory signaling molecule is 4-1BB. Incertain embodiments, the co-stimulatory signaling region of the CCRcomprises two co-stimulatory signaling molecules. In certainembodiments, the two co-stimulatory signaling molecules are CD28 and4-1BB. A second antigen is selected so that expression of both the firstantigen and the second antigen is restricted to the targeted cells(e.g., cancerous tissue or cancerous cells). Similiar to a CAR, theextracellular antigen-binding domain can be a scFv, a Fab, a F(ab)2, ora fusion protein with a heterologous sequence to form the extracellularantigen-binding domain.

In certain embodiments, the CCR is co-expressed with an antigenrecognizing receptor (e.g. CAR or TCR) binding to an antigen that isdifferent from the antigen to which the CCR binds, e.g., the antigenrecognizing receptor binds to a first antigen and the CCR binds to asecond antigen. In certain embodiments, the antigen recognizing receptoris a CAR. In certain embodiments, the immunoresponsive cell expressingthe antigen recognizing receptor (e.g., CAR) and the CCR exhibits agreater degree of cytolytic activity against cells that are positive forboth the first antigen and the second antigen as compared to againstcells that are singly positive for the first antigen. In certainembodiments, the immunoresponsive cell expressing the antigenrecognizing receptor (e.g., CAR) and the CCR exhibits substantially noor negligible cytolytic activity against cells that are singly positivefor the first antigen.

In certain embodiments, the antigen recognizing receptor (e.g., CAR) isnot potent or efficient, e.g., an antigen recognizing receptor (e.g., aCAR) that exhibits substantially no or negligible cytolytic activityagainst cells that are singly positive for the antigen to which theantigen recognizing receptor binds. In certain embodiments, the antigenrecognizing receptor (e.g., CAR) binds to the first antigen with a lowbinding affinity, e.g., a dissociation constant (K_(d)) of about 1×10⁻⁸M or more, about 5×10⁻⁸ M or more, about 1×10⁻⁷ M or more, about 5×10⁻⁷M or more, or about 1×10⁻⁶ M or more, or from about 1×10⁻⁸ M to about1×10⁻⁶ M. In certain embodiments, the binding affinity of a CAR refersto the binding affinity of the extracellular antigen-binding domain(e.g., scFv) of the CAR to the antigen. In certain embodiments, theantigen recognizing receptor (e.g., CAR) binds to the first antigen witha low binding avidity. In certain embodiments, the antigen recognizingreceptor (e.g., CAR) binds to the first antigen at an epitope of lowaccessibility. In certain embodiments, the antigen recognizing receptor(e.g., CAR) binds to the first antigen with a binding affinity that islower compared to the binding affinity with which the CCR binds to thesecond antigen. In certain embodiments, the CCR binds to the secondantigen with a binding affinity K_(d) of from about 1×10⁻⁹ M to about1×10⁻⁷ M, e.g., about 1×10⁻⁷ M or less, about 1×10⁻⁸M or less, or about1×10⁻⁹ M or less.

Tumor Microenvironment

Tumors have a microenvironment that is hostile to the host immuneresponse involving a series of mechanisms by malignant cells to protectthemselves from immune recognition and elimination. This “hostile tumormicroenvironment” comprises a variety of immune suppressive factorsincluding infiltrating regulatory CD4⁺ T cells (Tregs), myeloid derivedsuppressor cells (MDSCs), tumor associated macrophages (TAMs), immunesuppressive cytokines including IL-10 and TGF-β, and expression ofligands targeted to immune suppressive receptors expressed by activatedT cells (CTLA-4 and PD-1). These mechanisms of immune suppression play arole in the maintenance of tolerance and suppressing inappropriateimmune responses, however within the tumor microenvironment thesemechanisms prevent an effective anti-tumor immune response. Collectivelythese immune suppressive factors can induce either marked anergy orapoptosis of adoptively transferred CAR modified T cells upon encounterwith targeted tumor cells.

Myeloid Malignancies and Acute Myeloid Leukemia (AML)

Myeloid malignancies are clonal diseases caused by dysfunction ofhematopoietic stem cells or progenitor cells, resulting from genetic andepigenetic alterations that disrupt key processes such as cellproliferation and differentiation. Myeloid malignancies can be chronicor acute. Chronic diseases include myeloproliferative neoplasms (MPN),myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia(CMML). MPNs include chronic myeloid leukemia (CML) and non-CML MPNssuch as polycythemia vera (PV), essential thrombocythemia (ET) andprimary myelofibrosis (PMF). Acute diseases include acute myeloidleukemia (AML).

AML is characterized by the rapid growth of abnormal leukocytes whichaccumulate in the bone marrow and disrupt the production of normal bloodcells. Symptoms of AML include fatigue, shortness of breath, increasedsusceptibility of infection, and easy bruising and bleeding. Themajority of AML cases occur de novo, but some cases can be secondary toa chronic disease. There are eight different subtypes of AML:myeloblastic—undifferentiated (M0), myeloblastic—minimal maturation(M1), myeloblastic—full maturation (M2), promyeloctic (M3),myelomonocytic (M4), monocytic (M5), erythroleukemia (M6), andmegakaryocytic (M7). The classification is based on the type of cellfrom which the leukemia is originated and how mature the cells are.

AML Antigens

Antigens suitable for CAR targets to AML have been reported: 1) Lewis(Le)-Y, a difucosylated carbohydrate antigen, targeted in a phase Istudy of four patients with relapsed AML. Infusion of second generationCD28-based CARs resulted in stable/transient remission of threepatients, who ultimately progressed, despite T cell persistence (Ritchieet al., 2013); 2) CD123, the high-affinity interleukin-3 receptorα-chain; a partial remission was induced in a patient with FLT3-ITD+ AMLtreated with a third generation CD123-CD28/CD137/CD27/CD3z/iCaps9 CAR(YiLuo, 2015). Preclinical studies resulted in significant myeloablation(Gill et al., 2014); 3) CD33 is a myeloid-specific sialic acid-bindingreceptor, also targeted by gentuzumab ozogamicin (GO)(Administration,2010), with demonstrated survival benefit (Hills et al., 2014; Ravandiet al., 2012). Preclinical activity of CD33 CAR⁺ CIK cells resulted inslowing disease progression (Pizzitola et al., 2014) and CD33 CAR⁺ Tshowed significant effector functions in vitro and in vivo withreduction of myeloid progenitors (Kenderian et al., 2015). One AMLpatient was treated with CD33 CAR T cells at the Chinese PLA GeneralHospital, showing transient efficacy and mild fluctuations in bilirubin(Wang et al., 2015) and a clinical trial is registered as NCT01864902;4). Folate receptor β is a myeloid-lineage antigen (Lynn et al., 2016;Lynn et al., 2015). However, none of these meet the criteria of an idealCAR target.

The present disclosure provides new AML antigens suitable for CARtargets, which include EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF, CPM,ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1, MRP1,ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4,LILRB4, LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ,SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13,LILRB2, EMB, CD96, LILRB3, LILRA6, LILRA2, and SLC19A1.

ADGRE2/EMR2

EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2)(official name: adhesion G protein-coupled receptor E2 gene (ADGRE2),GenBank ID: 30817, also known as VBU and CD312) is a gene encoding amember of G-protein coupled receptors, and is expressed mainly inmyeloid cells where it promotes cell-cell adhesion through interactionwith chondroitin sulfate chains.

CD33

CD33 (GenBank ID: 945, also known as Siglec-3, sialic acid bindingIg-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67) is a transmembranereceptor expressed on cells of myeloid lineage. It is mainly expressedin myeloid cells.

IL10RB

Interleukin 10 receptor subunit beta (GenBank ID: 3588, also known asCRFB4; CRF2-4; D21S58; D21S66; CDW210B; and IL-10R2) is a gene encodinga cytokine receptor, which is an accessory chain essential for theactive interleukin 10 receptor complex.

PLXNC1

Plexin C1 (GenBank ID: 10154, also known as CD232; VESPR; and PLXN-C1)is a gene encoding a member of the plexin family, which aretransmembrane receptors for semaphorins.

PIEZO1

Piezo type mechanosensitive ion channel component 1 (GenBank ID: 9780,also known as DHS; Mib; LMPH3; and FAM38A) is a gene encoding amechanically-activated ion channel that links mechanical forces tobiological signals.

CD300LF

CD300 molecule like family member f (GenBank ID: 146722, also known asCLM1; NKIR; CLM-1; IREM1; LMIR3; CD300f; IREM-1; and IgSF13) is a geneencoding a member of the CD300 protein family, which are cell surfaceglycoproteins with a single IgV-like extracellular domain.

CPM

carboxypeptidase M (GenBank ID: 1368) is a gene encoding amembrane-bound arginine/lysine carboxypeptidase.

ITFG3

Integrin alpha FG-GAP repeat containing 3 (official name: family withsequence similarity 234 member A (FAM234A), GenBank ID: 83986, alsoknown as gs19, and C16orf9) is a gene encoding a member of proteinscontaining integrin alpha FG-GAP repeat.

TTYH3

Tweety family member 3 (GenBank ID: 80727) is a gene encoding a memberof the chloride anion channels.

ITGA4

Integrin subunit alpha 4 (GenBank ID: 3676, also known as IA4 and CD49D)is a gene encoding a member of the integrin alpha chain family ofproteins.

SLC9A1

Solute carrier family 9 member A1 (GenBank ID: 6548, also known as APNH;NHE1; LIKNS; NHE-1; and PPP1R143) is a gene encoding a Na+/H+antiporter.

MBOAT7

Membrane bound O-acyltransferase domain containing 7 (GenBank ID: 79143,also known as BB1; LRC4; LENG4; LPIAT; MBOA7; OACT7; and hMBOA-7) is agene encoding a member of the membrane-bound O-acyltransferases familyof integral membrane proteins that have acyltransferase activity.

CD38

CD38 (GenBank ID: 952, also known as ADPRC1 and ADPRC1) is a geneencoding a non-lineage-restricted, type II transmembrane glycoproteinthat synthesizes and hydrolyzes cyclic adenosine 5′-diphosphate-ribose,an intracellular calcium ion mobilizing messenger.

SLC6A6

Solute carrier family 6 member 6 (GenBank ID: 6533, also known as TAUT)is a gene encoding a member of a family of sodium and chloride-iondependent transporters.

ENG

Endoglin (GenBank ID: 2022, also known as END; HHT1; and ORW1) is a geneencoding a homodimeric transmembrane protein which is a majorglycoprotein of the vascular endothelium.

SIRPB1

Signal regulatory protein beta 1 (GenBank ID: 10326, also known asCD172b and SIRP-BETA-1) is a gene encoding a member of thesignal-regulatory-protein (SIRP) family, and also belongs to theimmunoglobulin superfamily.

MRP1

Multidrug resistance-associated protein 1 (MRP1) (official name: ATPbinding cassette subfamily C member 1 (ABCC1), GenBank ID: 4363, alsoknown as MRP; ABCC; GS-X; and ABC29) is a gene encoding a member of thesuperfamily of ATP-binding cassette (ABC) transporters.

ITGA5

Integrin subunit alpha 5 (GenBank ID: 3678, also known as FNRA; CD49e;VLA-5; and VLA5A) is a gene encoding a member of the integrin alphachain family of proteins.

SLC43A3

Solute carrier family 43 member 3 (GenBank ID: 29015, also known asEEG1; FOAP-13; PRO1659; and SEEEG-1) is a gene encoding an equilibrativenucleobase transporter.

MYADM

Myeloid associated differentiation marker (GenBank ID: 91663, also knownas SB135) is a gene encoding a protein highly up-regulated asmultipotent progenitor cells differentiate into myeloid cells. Theprotein is predicted to be a membrane protein.

ICAM1

Intercellular adhesion molecule 1 (GenBank ID: 3383, also known as BB2;CD54; and P3.58) is a gene encoding a cell surface glycoprotein.

SLC44A1

Solute carrier family 44 member 1 (GenBank ID: 23446, also known asCD92; CTL1; CDW92; and CHTL1) is a gene encoding a choline transporterwith an intermediate affinity for choline.

CCR1

C-C motif chemokine receptor 1 (GenBank ID: 1230, also known as CKR1;CD191; CKR-1; HM145; CMKBR1; MIP1 aR; and SCYAR1) is a gene encoding amember of the beta chemokine receptor family, which is predicted to be aseven transmembrane protein similar to G protein-coupled receptors.

SLC22A5

Solute carrier family 22 member 5 (GenBank ID: 6584, also known as CDSPand OCTN2) is a gene encoding a plasma integral membrane protein thatfunctions as an organic cation transporter. The protein also functionsas a sodium-dependent high affinity carnitine transporter, which isinvolved in active cellular uptake of carnitine.

TFR2

Transferrin receptor 2 (GenBank ID: 7036, also known as HFE3; and TFRC2)is a gene encoding a single-pass type II membrane protein, which is amember of the transferrin receptor-like family.

KCNN4

Potassium calcium-activated channel subfamily N member 4 (GenBank ID:3783, also known as IK; IK1; SK4; DHS2; KCA4; hSK4; IKCA1; hKCa4;KCa3.1; and hIKCa1) is a gene encoding a part of a potentiallyheterotetrameric voltage-independent potassium channel that is activatedby intracellular calcium.

LILRB4

Leukocyte immunoglobulin like receptor B4 (GenBank ID: 11006, also knownas ILT3; LIR5; CD85K; ILT-3; and LIR-5) is a gene encoding a member ofthe leukocyte immunoglobulin-like receptor (LIR) family.

LTB4R

Leukotriene B4 receptor (GenBank ID: 1241, also known as BLT1; BLTR;P2Y7; GPR16; LTBR1; P2RY7; CMKRL1; and LTB4R1) is a gene encoding amember of leukotriene receptors, which are G protein-coupled receptorsthat bind and are activated by the leukotrienes.

CD70

CD70 (GenBank ID: 970, also known as CD27L; CD27LG; TNFSF7; and TNLG8A)is a gene encoding a cytokine that belongs to the tumor necrosis factor(TNF) ligand family.

GYPA

Glycophorin A (MNS blood group) (GenBank ID: 2993, also known as MN;GPA; MNS; GPSAT; PAS-2; CD235a; GPErik; HGpMiV; HGpMiXI; and HGpSta(C))is a gene encoding a major sialoglycoprotein of the human erythrocytemembrane which bear the antigenic determinants for the MN and Ss bloodgroups.

FCGR1A

Fc fragment of IgG receptor Ia (GenBank ID: 2209, also known as CD64;FCRI; CD64A; and IGFR1) is a gene encoding a high-affinity Fc-gammareceptor.

LILRB2

Leukocyte immunoglobulin like receptor B2 (GenBank ID: 10288, also knownas CD85d; ILT4; LIR2; CD85D; ILT-4; LIR-2; MIR10; and MIR-10) is a geneencoding a member of the leukocyte immunoglobulin-like receptor (LIR)family. The encoded protein is expressed on myeloid and B cells, actingto suppress the immune response. It is also expressed on NSCLC cells(Sun et al., 2008).

CLEC12A

C-type lectin domain family 12 member A (GenBank ID: 160364, also knownas CLL1; MICL; CD371; CLL-1; and DCAL-2) is a gene encoding a member ofthe C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily.Members of this family share a common protein fold and have diversefunctions, such as cell adhesion, cell-cell signaling, glycoproteinturnover, and roles in inflammation and immune response.

CD123

Interleukin 3 receptor subunit alpha (GenBank ID: 3563, also known asIL3R; CD123; IL3RX; IL3RY; IL3RAY; and hIL-3Ra) is a gene encoding aninterleukin 3 specific subunit of a heterodimeric cytokine receptor. Thereceptor is comprised of a ligand specific alpha subunit and a signaltransducing beta subunit shared by the receptors for interleukin 3(IL3), colony stimulating factor 2 (CSF2/GM-CSF), and interleukin 5(IL5).

ITGB5

Integrin subunit beta 5 (GenBank ID: 3693) is a gene encoding a memberof the integrin beta chain family of proteins.

PTPRJ

Protein tyrosine phosphatase, receptor type J (GenBank ID: 5795, alsoknown as DEP1; SCC1; CD148; HPTPeta; and R-PTP-ETA) is a gene encoding amember of the protein tyrosine phosphatase (PTP) family. PTPs are knownto be signaling molecules that regulate a variety of cellular processes,including cell growth, differentiation, mitotic cycle, and oncogenictransformation.

SLC30A1

Solute carrier family 30 member 1 (GenBank ID: 7779, also known as ZNT1and ZRC1) is a gene encoding a zinc transporter.

EMC10

ER membrane protein complex subunit 10 (GenBank ID: 284361, also knownas HSM1; HSS1; and C19orf63) is a gene encoding a component of the ERmembrane protein complex (EMC) in mammals.

TNFRSF1B

TNF receptor superfamily member 1B (GenBank ID: 7133, also known as p75;TBPII; TNFBR; TNFR2; CD120b; TNFR1B; TNFR80; TNF-R75; p75TNFR; andTNF—R-II) is a gene encoding a member of the TNF-receptor superfamily.This protein and TNF-receptor 1 form a heterocomplex that mediates therecruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, whichpossess E3 ubiquitin ligase activity.

CD82

CD82 molecule (GenBank ID: 3732, also known as R2; 4F9; C33; IA4; ST6;GR15; KAI1; SAR2; and TSPAN27) is a gene encoding a membraneglycoprotein that is a member of the transmembrane 4 superfamily.

ITGAX

Integrin subunit alpha X (GenBank ID: 3687, also known as CD11C andSLEB6) is a gene encoding a member of the integrin alpha chain family ofproteins. This protein combines with the beta 2 chain (ITGB2) to form aleukocyte-specific integrin referred to as inactivated-C3b (iC3b)receptor 4 (CR4).

CR1

Complement C3b/C4b receptor 1 (GenBank ID: 1378, also known as KN; C3BR;C4BR; and CD35) is a gene encoding a member of the receptors ofcomplement activation (RCA) family and is located in the ‘cluster RCA’region of chromosome 1, which is a monomeric single-pass type I membraneglycoprotein found on erythrocytes, leukocytes, glomerular podocytes,and splenic follicular dendritic cells.

DAGLB

Diacylglycerol lipase beta (GenBank ID: 221955, also known as KCCR13Land DAGLBETA) is a gene encoding an enzyme in the biosynthesis of theendocannabinoid 2-arachidonoylglycerol, which catalyzes the hydrolysisof diacylglycerol.

SEMA4A

Semaphorin 4A (GenBank ID: 64218, also known as RP35; SEMB; SEMAB; andCORD10) is a gene encoding a member of the semaphorin family of solubleand transmembrane proteins, which is a single-pass type I membraneprotein containing an immunoglobulin-like C2-type domain, a PSI domainand a sema domain.

TLR2

Toll like receptor 2 (GenBank ID: 7097, also known as TIL4 and CD282) isa gene encoding a member of the Toll-like receptor (TLR) family whichplays a fundamental role in pathogen recognition and activation ofinnate immunity. This protein is a cell-surface protein that can formheterodimers with other TLR family members to recognize conservedmolecules derived from microorganisms known as pathogen-associatedmolecular patterns (PAMPs).

P2RY13

Purinergic receptor P2Y13 (GenBank ID: 53829, also known as GPCR1;GPR86; GPR94; P2Y13; SP174; and FKSG77) is a gene encoding a member ofthe family of G-protein coupled receptors. This receptor is activated byADP.

EMB

Embigin (GenBank ID: 133418, also known as GP70) is a gene encoding atransmembrane glycoprotein that is a member of the immunoglobulinsuperfamily.

CD96

CD96 molecule (GenBank ID: 10225) is a gene encoding a member of theimmunoglobulin superfamily.

LILRB3

Leukocyte immunoglobulin like receptor B3 (GenBank ID: 11025, also knownas HL9; ILT5; LIR3; PIRB; CD85A; ILT-5; LIR-3; PIR-B; and LILRA6) is agene encoding a member of the leukocyte immunoglobulin-like receptor(LIR) family, which is found in a gene cluster at chromosomal region19q13.4. The encoded protein belongs to the subfamily B class of LIRreceptors which contain two or four extracellular immunoglobulindomains, a transmembrane domain, and two to four cytoplasmicimmunoreceptor tyrosine-based inhibitory motifs (ITIMs).

LILRA6

Leukocyte immunoglobulin like receptor A6 (GenBank ID: 79168, also knownas ILT5; ILT8; CD85b; ILT-8; LILRB3; and LILRB6) is a gene encoding amember of a family of immunoreceptors that are expressed predominantlyon monocytes and B cells, and at lower levels on dendritic cells andnatural killer cells.

LILRA2

Leukocyte immunoglobulin like receptor A2 (GenBank ID: 11027, also knownas ILT1; LIR7; CD85H; LIR-7) is a gene encoding a member of a family ofimmunoreceptors that are expressed predominantly on monocytes and Bcells, and at lower levels on dendritic cells and natural killer cells.

In certain embodiments, the antigen suitable for antigen recognizingreceptor (CAR or TCR) and/or CCR targets for treating AML is selectedfrom the group consisting of EMR2, CD33, IL10RB, PLXNC1, PIEZO1,CD300LF, CPM, ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG,SIRPB1, MRP1, ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5,TFR2, KCNN4, LILRB4, LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5,PTPRJ, SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2,P2RY13, LILRB2, EMB, CD96, LILRB3, LILRA6, LILRA2, and SLC19A1.

Immunoresponsive Cells

The presently disclosed subject matter provides cells comprising anantigen recognizing receptor (e.g., CAR or TCR) targeting an antigen ofinterest, e.g., an AML antigen, and methods of using such cells fortreating myeloid disorders. For example, a T cell comprsing a chimericantigen receptor that recognizes EMR2. Such cells are administered to ahuman subject in need thereof for treating and/or preventing myeloiddisorders.

The immunoresponsive cells of the presently disclosed subject matter canbe cells of the lymphoid lineage. The lymphoid lineage, comprising B, Tand natural killer (NK) cells, provides for the production ofantibodies, regulation of the cellular immune system, detection offoreign agents in the blood, detection of cells foreign to the host, andthe like. Non-limiting examples of immunoresponsive cells of thelymphoid lineage include T cells, Natural Killer (NK) cells, embryonicstem cells, and pluripotent stem cells (e.g., those from which lymphoidcells may be differentiated). T cells can be lymphocytes that mature inthe thymus and are chiefly responsible for cell-mediated immunity. Tcells are involved in the adaptive immune system. The T cells of thepresently disclosed subject matter can be any type of T cells,including, but not limited to, T helper cells, cytotoxic T cells, memoryT cells (including central memory T cells, stem-cell-like memory T cells(or stem-like memory T cells), and two types of effector memory T cells:e.g., T_(EM) cells and T_(EMRA) cells, Regulatory T cells (also known assuppressor T cells), Natural killer T cells, Mucosal associatedinvariant T cells, and γδ T cells. Cytotoxic T cells (CTL or killer Tcells) are a subset of T lymphocytes capable of inducing the death ofinfected somatic or tumor cells. A patient's own T cells may begenetically modified to target specific antigens through theintroduction of an antigen recognizing receptor, e.g., a CAR or a TCR.

Natural killer (NK) cells can be lymphocytes that are part ofcell-mediated immunity and act during the innate immune response. NKcells do not require prior activation in order to perform theircytotoxic effect on target cells.

Types of human lymphocytes of the presently disclosed subject matterinclude, without limitation, peripheral donor lymphocytes geneticallymodified to express CARs (Sadelain, M., et al. 2003 Nat Rev Cancer3:35-45), peripheral donor lymphocytes genetically modified to express afull-length antigen-recognizing T cell receptor complex comprising the αand β heterodimer (Morgan, R. A., et al. 2006 Science 314:126-129),lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs)in tumor biopsies (Panelli, M. C., et al. 2000 J Immunol 164:495-504;Panelli, M. C., et al. 2000 J Immunol 164:4382-4392), and selectively invitro-expanded antigen-specific peripheral blood leukocytes employingartificial antigen-presenting cells (AAPCs) or pulsed dendritic cells(Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, G. A.,et al. 2003 Blood 102:2498-2505). The T cells may be autologous,allogeneic, or derived in vitro from engineered progenitor or stemcells.

The unpurified source of CTLs may be any known in the art, such as thebone marrow, fetal, neonate or adult or other hematopoietic cell source,e.g., fetal liver, peripheral blood or umbilical cord blood. Varioustechniques can be employed to separate the cells. For instance, negativeselection methods can remove non-CTLs initially. mAbs are particularlyuseful for identifying markers associated with particular cell lineagesand/or stages of differentiation for both positive and negativeselections.

A large proportion of terminally differentiated cells can be initiallyremoved by a relatively crude separation. For example, magnetic beadseparations can be used initially to remove large numbers of irrelevantcells. Preferably, at least about 80%, usually at least 70% of the totalhematopoietic cells will be removed prior to cell isolation.

Procedures for separation include, but are not limited to, densitygradient centrifugation; resetting; coupling to particles that modifycell density; magnetic separation with antibody-coated magnetic beads;affinity chromatography; cytotoxic agents joined to or used inconjunction with a mAb, including, but not limited to, complement andcytotoxins; and panning with antibody attached to a solid matrix, e.g.plate, chip, elutriation or any other convenient technique.

Techniques for separation and analysis include, but are not limited to,flow cytometry, which can have varying degrees of sophistication, e.g.,a plurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels.

The cells can be selected against dead cells, by employing dyesassociated with dead cells such as propidium iodide (PI). Preferably,the cells are collected in a medium comprising 2% fetal calf serum (FCS)or 0.2% bovine serum albumin (BSA) or any other suitable, preferablysterile, isotonic medium.

In certain embodiments, an allogenic immunoresponsive cell (e.g., anallogenic T cell) is used. In certain embodiments, a universal T cellwith deficient TCRaPαβ is used. The methods of developing universal Tcells are described in the art, for example, in Valton et al., MolecularTherapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012119:5697-5705, which are incorporated by reference in their entireties.

In certain embodiments, the presently disclosed subject matter providesan isolated immunoresponsive cell comprising an antigen recognizingreceptor (e.g. CAR or TCR) that binds to an antigen selected from thegroup consisting of EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF, CPM,ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1, MRP1,ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4,LILRB4, LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ,SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13,LILRB2, EMB, CD96, LILRB3, LILRA6, LILRA2, and SLC19A1. In certainembodiments, the antigen is selected from the group consisting of LTB4R,EMR2, CD33, MYADM, PIEZO1, SIRPB1, SLC9A1, KCNN4, ENG, ITGA5, and CD70.In certain embodiments, the antigen is selected from the groupconsisting of LTB4R, EMR2, MYADM and PIEZO1. In certain embodiments, theantigen is selected from the group consisting of CD82, TNFRSF1B, EMR2,ITGB5, CCR1, CD96, PTPRJ, CD70 and LILRB2. In certain embodiments, theantigen is selected from the group consisting of TNFRSF1B, EMR2, CCR1,CD96, CD70 and LILRB2. In certain embodiments, the antigen is selectedfrom the group consisting of EMR2, CCR1, CD70 and LILRB2. In certainnon-limiting embodiments, the antigen is EMR2. In certain embodiments,the binding of the antigen recognizing receptor to the antigen iscapable of activating the immunoresponsive cell.

In certain embodiments, the presently disclosed subject matter providesan isolated immunoresponsive cell comprising: (a) an antigen recognizingreceptor (e.g. CAR or TCR) that binds to a first antigen, whereinbinding of the receptor to the first antigen is capable of activatingthe immunoresponsive cell, and (b) a chimeric co-stimulating receptor(CCR) that binds to a second antigen, wherein binding of the CCR to thesecond antigen is capable of stimulating the immunoresponsive cell,wherein each of the first and second antigens is selected from the groupconsisting of EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF, CPM, ITFG3,TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1, MRP1, ITGA5,SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4, LILRB4,LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ, SLC30A1, EMC10,TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13, LILRB2, EMB,CD96, LILRB3, LILRA6, LILRA2, and SLC19A1, and wherein the first antigenand the second antigen are different. In certain embodiments, the firstantigen and the second antigen are a combination selected from the groupconsisting of LTB4R and EMR2, LTB4R and CD33, LTB4R and ENG, LTB4R andMYADM, LTB4R and PIEZO1, LTB4R and SIRPB1, LTB4R and SLC9A1, LTB4R andITGA5, LTB4R and CD70, LTB4R and KCNN4. EMR2 and CD33, EMR2 and ENG,EMR2 and MYADM, EMR2 and PIEZO1, EMR2 and SIRPB1, EMR2 and SLC9A1, EMR2and ITGA5, EMR2 and CD70, EMR2 and KCNN4, CD33 and ENG, CD33 and MYADM,CD33 and PIEZO1, CD33 and SIRPB1, CD33 and SLC9A1, CD33 and ITGA5, CD33and CD70, CD33 and KCNN4, ENG and MYADM, ENG and PIEZO1, ENG and SIRPB1,ENG and SLC9A1, ENG and ITGA5, ENG and CD70, ENG and KCNN4, MYADM andPIEZO1, MYADM and SIRPB1, MYADM and SLC9A1, MYADM and ITGA5, MYADM andCD70, MYADM and KCNN4, PIEZO1 and SIRPB1, PIEZO1 and SLC9A1, PIEZO1 andITGA5, PIEZO1 and CD70, PIEZO1 and KCNN4, SIRPB1 and SLC9A1, SIRPB1 andITGA5; SIRPB1 and CD70; SIRPB1 and KCNN4, SLC9A1 and ITGA5, SLC9A1 andCD70, SLC9A1 and KCNN4, ITGA5 and CD70, ITGA5 and KCNN4, CD70 and KCNN4,EMR2 and CD33, CCR1 and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A, EMR2and CLEC12A, EMR2 and CD96, CCR1 and CD33, CCR1 and CD96, CD70 andCLEC12A, CD70 and CD96, LILRB2 and CD33, LILRB2 and CD96, and EMR2 andCD70. In certain embodiments, the first antigen and the second antigenare a combination selected from the group consisting of EMR2 and CD33,CCR1 and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A, LTB4R1 and CD70,CD70 and EMR2, and LTB4R1 and EMR2.

In certain embodiments, the immunoresponsive cell exhibits a greaterdegree of cytolytic activity against cells that are positive for boththe first antigen and the second antigen as compared to against cellsthat are singly positive for the first antigen. In certain embodiments,the antigen recognizing receptor comprises an antigen recognizingreceptor (e.g. CAR or TCR) that binds to a first antigen with a lowbinding affinity or a low binding avidity. In certain embodiments, theantigen recognizing receptor (e.g. CAR or TCR) binds to the firstantigen at an epitope of low accessibility. In certain embodiments, theantigen recognizing receptor (e.g. CAR or TCR) binds to the firstantigen with a binding affinity that is lower compared to the bindingaffinity with which the CCR binds to the second antigen. In non-limitingembodiments herein, for example certain embodiments that employ aCAR/CCR combination, the CCR-recognized antigen is used to directcostimulation to enhance or rescue suboptimal function of a CAR or TCRtargeting a second antigen. Using this approach, the immunoresponsive Tcells are more restricted to dual-antigen positive tumor cells, thusrelaxing the expression criteria for at least one of the pairedantigens; however the presence of the CAR or TCR antigen becomes moreimportant to avert antigen escape. In contrast, immunoresponsive cellsexpressing a CAR/CAR or CAR/TCR combination would engage tissuesexpressing either antigen alone and depending on choice of antigen,binding affinity, and functionality, could avoid undesirable off-targeteffects. Thus, with regards to increasing therapeutic efficacy, thefirst principle for choosing target antigen is to maximize the number oftargetable tumor cells, addressing the challenge of clonalheterogeneity. Another priority is to target leukemia stem cells(“LSCs”) to achieve satisfactory therapeutic benefit. Finally, pairingchoices should favor redundant expression of the two targets in thetumor in order to minimize the risk of antigen escape. Accordingly, innon-limiting embodiments, provided herein are an immunoresponsive cellthat comprises (i) a first antigen recognizing receptor (e.g. CAR orTCR) that binds to a first antigen and (ii) a second antigen recognizingreceptor (e.g. CAR or TCR) that binds to a second antigen, wherein thecombination of both receptors binding to their targets produces atherapeutic effect. In certain non-limiting embodiments, binding to onlyone target does not achieve a therapeutic effect. For example, the firstand second antigen recognizing receptor can both be CARs; alternatively,the first antigen recognizing receptor can be a CAR and the secondantigen binding receptor can be a TCR, or the first antigen recognizingreceptor can be a TCR and the second antigen recognizing receptor can bea CAR, or both antigen recognizing receptors can be TCRs. Optionally,said immunoresponsive cell may further comprise a third antigentargeting molecule, which may be a CAR, TCR, or CCR that recognizes athird antigen. In non-limiting embodiments, the first, second, andoptional third antigen are different. In non-limiting embodiments, eachof the first antigen, second antigen and third antigen is selected fromthe group consisting of EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF,CPM, ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1,MRP1, ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4,LILRB4, LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ,SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13,LILRB2, EMB, CD96, LILRB3, LILRA6, LILRA2, and SLC19A1, and the firstantigen and the second antigen are different. In certain embodiments,the first antigen and the second antigen are a combination selected fromthe group consisting of LTB4R and EMR2, LTB4R and CD33, LTB4R and ENG,LTB4R and MYADM, LTB4R and PIEZO1, LTB4R and SIRPB1, LTB4R and SLC9A1,LTB4R and ITGA5, LTB4R and CD70, LTB4R and KCNN4. EMR2 and CD33, EMR2and ENG, EMR2 and MYADM, EMR2 and PIEZO1, EMR2 and SIRPB1, EMR2 andSLC9A1, EMR2 and ITGA5, EMR2 and CD70, EMR2 and KCNN4, CD33 and ENG,CD33 and MYADM, CD33 and PIEZO1, CD33 and SIRPB1, CD33 and SLC9A1, CD33and ITGA5, CD33 and CD70, CD33 and KCNN4, ENG and MYADM, ENG and PIEZO1,ENG and SIRPB1, ENG and SLC9A1, ENG and ITGA5, ENG and CD70, ENG andKCNN4, MYADM and PIEZO1, MYADM and SIRPB1, MYADM and SLC9A1, MYADM andITGA5, MYADM and CD70, MYADM and KCNN4, PIEZO1 and SIRPB1, PIEZO1 andSLC9A1, PIEZO1 and ITGA5, PIEZO1 and CD70, PIEZO1 and KCNN4, SIRPB1 andSLC9A1, SIRPB1 and ITGA5, SIRPB1 and CD70, SIRPB1 and KCNN4, SLC9A1 andITGA5, SLC9A1 and CD70, SLC9A1 and KCNN4, ITGA5 and CD70, ITGA5 andKCNN4, CD70 and KCNN4, EMR2 and CD33, CCR1 and CLEC12A, CD70 and CD33,LILRB2 and CLEC12A, EMR2 and CLEC12A, EMR2 and CD96, CCR1 and CD33, CCR1and CD96, CD70 and CLEC12A, CD70 and CD96, LILRB2 and CD33, LILRB2 andCD96, EMR2 and CD70. In certain embodiments, the first antigen and thesecond antigen are a combination selected from the group consisting ofEMR2 and CD33, CCR1 and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A,LTB4R1 and CD70, CD70 and EMR2, and LTB4R1 and EMR2 In addition, innon-limiting embodiments, where an antigen recognizing receptor is aTCR, a target antigen can be WT1 or PRAME. In certain non-limitingembodiments, the first antigen is EMR2. In certain embodiments, theimmunoresponsive cell exhibits a greater degree of cytolytic activityagainst cells that are positive for both the first antigen and thesecond antigen as compared to against cells that are singly positive forthe first antigen. In certain embodiments, the first antigen recognizingreceptor comprises an antigen recognizing receptor (e.g. CAR or TCR)that binds to a first antigen with a low binding affinity or a lowbinding avidity. In certain embodiments, the first antigen recognizingreceptor (e.g. CAR or TCR) binds to the first antigen at an epitope oflow accessibility. In certain embodiments, the first antigen recognizingreceptor (e.g. CAR or TCR) binds to the first antigen with a bindingaffinity that is lower compared to the binding affinity with which thesecond antigen recognizing receptor binds to the second antigen. Incertain embodiments, the first antigen recognizing receptor (e.g. CAR orTCR) binds to the first antigen with a binding affinity that is at least5 fold lower compared to the binding affinity with which the secondantigen recognizing receptor binds to the second antigen. In certainembodiments, the first antigen recognizing receptor (e.g. CAR or TCR)binds to the first antigen with a binding affinity that is at leat 10fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, 100 fold, 200 fold, 5000 fold, 1000 fold, 5000 fold, or 10000 foldlower compared to the binding affinity with which the second antigenrecognizing receptor binds to the second antigen.

In certain non-limiting embodiments, an immunoresponsive cell comprisestwo CAR constructs. In certain embodiments, the cell comprises a firstCAR comprising a first intracellular signaling domain, and a second CARcomprising a second introcellular signalling domain, wherein the firstintracellular signaling domain and the second intracellular signalingdomain are different. In certain embodiments, each of the firstintracellular signaling domain and the second intracellular signalingdomain is selected from the group consisting of CD3ζ-chain, CD97,CD11a-CD18, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28 signalingdomain, or combinations thereof, wherein the first intracellularsignaling domain and the second intracellular signaling domain aredifferent. In certain embodiments, each of the first intracellularsignaling domain and the second intracellular signaling domain comprisesa CD3ζ-chain, and optionally further comprise a signaling domainselected from the group consisting of CD97, CD11a-CD18, CD2, ICOS, CD27,CD154, CD8, OX40, 4-1BB, CD28 signaling domain, or combinations thereof,wherein the first intracellular signaling domain and the secondintracellular signaling domain are different. In certain embodiments,the first intracellular signaling domain comprises a CD3ζ-chain and aCD28 signaling domain, and the second intracellular signaling domaincomprises a CD3ζ-chain. In certain embodiments, the first intracellularsignaling domain comprises a CD3ζ-chain and a CD28 signaling domain, andthe second intracellular signaling domain comprises a CD3ζ-chain and a4-1BB signaling domain. In certain embodiments, the first intracellularsignaling domain comprises a CD3ζ-chain and a 4-1BB signaling domain,and the second intracellular signaling domain comprises a CD3ζ-chain.

In certain non-limiting embodiments, an immunoresponsive cell maycomprise three elements; for example three species of CAR, or twospecies of CAR and one CCR; or two species of CAR and one TCR; or oneCAR, one TCR, and one CCR. Still further combinations adding additionalCAR, CCR, and/or TCR are provided.

In particular non-limiting embodiments, an immunoresponsive cell, suchas a T cell or NK cell, may comprise a CAR that specifically binds toCLEC12A, a CAR that specifically binds to CD70, and a CCR thatspecifically binds to ADGRE2.

In particular non-limiting embodiments, an immunoresponsive cell, suchas a T cell or NK cell, may comprise a CAR that specifically binds toCLEC12A, a CAR that specifically binds to CD70, and a CCR thatspecifically binds to CD33.

In particular non-limiting embodiments, an immunoresponsive cell, suchas a T cell or NK cell, may comprise a CAR that specifically binds toCLEC12A, a CCR that specifically binds to CD70, and a CAR thatspecifically binds to TIM3. A presently disclosed immunoresponsive cellcan further include at least one recombinant or exogenous co-stimulatoryligand. For example, a presently disclosed immunoresponsive cell can befurther transduced with at least one co-stimulatory igand, such that theimmunoresponsive cell co-expresses or is induced to co-express the AMLantigen-targeted CAR or TCR and the at least one co-stimulatory ligand.The interaction between the CAR and at least one co-stimulatory ligandprovides a non-antigen-specific signal important for full activation ofan immunoresponsive cell (e.g., T cell). Co-stimulatory ligands include,but are not limited to, members of the tumor necrosis factor (TNF)superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is acytokine involved in systemic inflammation and stimulates the acutephase reaction. Its primary role is in the regulation of immune cells.Members of TNF superfamily share a number of common features. Themajority of TNF superfamily members are synthesized as type IItransmembrane proteins (extracellular C-terminus) containing a shortcytoplasmic segment and a relatively long extracellular region. TNFsuperfamily members include, without limitation, nerve growth factor(NGF), CD40L (CD40L)/CD154, CD137L/4-1BBL, TNF-α, CD134L/OX40L/CD252,CD27L/CD70, Fas ligand (FasL), CD30L/CD153, tumor necrosis factor beta(TNFβ)/lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257/Bcell-activating factor (BAFF)/Blys/THANK/Tall-1, glucocorticoid-inducedTNF Receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand(TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a largegroup of cell surface and soluble proteins that are involved in therecognition, binding, or adhesion processes of cells. These proteinsshare structural features with immunoglobulins—they possess animmunoglobulin domain (fold). Immunoglobulin superfamily ligandsinclude, but are not limited to, CD80 and CD86, both ligands for CD28,PD-L1/(B7-H1) that ligands for PD-1. In certain embodiments, the atleast one co-stimulatory ligand is selected from the group consisting of4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinationsthereof. In certain embodiments, the immunoresponsive cell comprises onerecombinant co-stimulatory ligand that is 4-1BBL. In certainembodiments, the immunoresponsive cell comprises two recombinantco-stimulatory ligands that are 4-1BBL and CD80. CARs comprising atleast one co-stimulatory ligand are described in U.S. Pat. No.8,389,282, which is incorporated by reference in its entirety.

Vectors

Genetic modification of immunoresponsive cells (e.g., T cells, CTLcells, NK cells) can be accomplished by transducing a substantiallyhomogeneous cell composition with a recombinant DNA construct.Preferably, a retroviral vector (either gamma-retroviral or lentiviral)is employed for the introduction of the DNA construct into the cell. Forexample, a polynucleotide encoding a receptor that binds an antigen(e.g., a tumor antigen, or a variant, or a fragment thereof), can becloned into a retroviral vector and expression can be driven from itsendogenous promoter, from the retrovirallong terminal repeat, or from apromoter specific for a target cell type of interest. Non-viral vectorsmay be used as well.

For initial genetic modification of the cells to provide antigenreceptors, a retroviral vector is generally employed for transduction,however any other suitable viral vector or non-viral delivery system canbe used. For genetic modification of the cells to provide cellscomprising a CAR and a CCR, retroviral gene transfer (transduction)likewise proves effective. The CAR and CCR can be constructed in asingle, multicistronic expression cassette, in multiple expressioncassettes of a single vector, or in multiple vectors. Examples ofelements which create polycistronic expression cassete include, but isnot limited to, various Internal Ribosome Entry Sites (IRES, e.g.,poliovirus IRES and encephalomyocarditis virus IRES) and 2A peptides(e.g., P2A, T2A, E2A and F2A peptides). Combinations of retroviralvector and an appropriate packaging line are also suitable, where thecapsid proteins will be functional for infecting human cells. Variousamphotropic virus-producing cell lines are known, including, but notlimited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437);PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP(Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464).Non-amphotropic particles are suitable too, e.g., particles pseudotypedwith VSVG, RD114 or GALV envelope and any other known in the art.

Possible methods of transduction also include direct co-culture of thecells with producer cells, e.g., by the method of Bregni, et al. (1992)Blood 80:1418-1422, or culturing with viral supernatant alone orconcentrated vector stocks with or without appropriate growth factorsand polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat.22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

Other transducing viral vectors can be used to express a antigenreceptor, a CAR, a CCR, and/or other compotents of the invention in animmunoresponsive cell. Preferably, the chosen vector exhibits highefficiency of infection and stable integration and expression (see,e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al.,Current Eye Research 15:833-844, 1996; Bloomer et al., Journal ofVirology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996;and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Otherviral vectors that can be used include, for example, adenoviral,lentiviral, and adena-associated viral vectors, vaccinia virus, a bovinepapilloma virus, or a herpes virus, such as Epstein-Barr Virus (alsosee, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990;Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al.,Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson,Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller etal., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviralvectors are particularly well developed and have been used in clinicalsettings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson etal., U.S. Pat. No. 5,399,346).

Non-viral approaches can also be employed for the expression of aprotein in cell. For example, a nucleic acid molecule can be introducedinto a cell by administering the nucleic acid in the presence oflipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am.J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al.,Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal ofBiological Chemistry 264:16985, 1989), or by micro-injection undersurgical conditions (Wolff et al., Science 247:1465, 1990). Othernon-viral means for gene transfer include transfection in vitro usingcalcium phosphate, DEAE dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell. Transplantation of normal genes into the affected tissues of asubject can also be accomplished by transferring a normal nucleic acidinto a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (orits descendants) are injected into a targeted tissue or are injectedsystemically. Recombinant receptors can also be derived or obtainedusing transposases or targeted nucleases (e.g. Zinc finger nucleases,meganucleases, or TALE nucleases). Transient expression may be obtainedby RNA electroporation.

cDNA expression for use in polynucleotide therapy methods can bedirected from any suitable promoter (e.g., the human cytomegalovirus(CMV), simian virus 40 (SV40), or metallothionein promoters), andregulated by any appropriate mammalian regulatory element or intron(e.g. the elongation factor 1a enhancer/promoter/intron structure). Forexample, if desired, enhancers known to preferentially direct geneexpression in specific cell types can be used to direct the expressionof a nucleic acid. The enhancers used can include, without limitation,those that are characterized as tissue- or cell-specific enhancers.Alternatively, if a genomic clone is used as a therapeutic construct,regulation can be mediated by the cognate regulatory sequences or, ifdesired, by regulatory sequences derived from a heterologous source,including any of the promoters or regulatory elements described above.

The resulting cells can be grown under conditions similar to those forunmodified cells, whereby the modified cells can be expanded and usedfor a variety of purposes.

Polypeptides and Analogs

Also included in the presently disclosed subject matter are a CD8, CD28,CD3ζ, 4-1BB, EMR2, CD33, IL10RB, PLXNC1, PIEZO1, CD300LF, CPM, ITFG3,TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6, ENG, SIRPB1, MRP1, ITGA5,SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5, TFR2, KCNN4, LILRB4,LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5, PTPRJ, SLC30A1, EMC10,TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2, P2RY13, LILRB2, EMB,CD96, LILRB3, LILRA6, LILRA2, and SLC19A1 polypeptides or fragmentsthereof that are modified in ways that enhance their activity whenexpressed in an immunoresponsive cell. The presently disclosed subjectmatterprovides methods for optimizing an amino acid sequence or nucleicacid sequence by producing an alteration in the sequence. Suchalterations may include certain mutations, deletions, insertions, orpost-translational modifications. The presently disclosed subject matterfurther includes analogs of any naturally-occurring polypeptide of theinvention. Analogs can differ from a naturally occurring polypeptide ofthe invention by amino acid sequence differences, by post—translationalmodifications, or by both. Analogs of the invention will generallyexhibit at least about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or moreidentity with all or part of a naturally-occurring amino, acid sequenceof the invention. The length of sequence comparison is at least 5, 10,15 or 20 amino acid residues, preferably at least 25, 50, or 75 aminoacid residues, and more preferably more than 100 amino acid residues.Again, in an exemplary approach to determining the degree of identity, aBLAST program may be used, with a probability score between e⁻³ ande⁻¹⁰⁰ indicating a closely related sequence. Modifications include invivo and in vitro chemical derivatization of polypeptides, e.g.,acetylation, carboxylation, phosphorylation, or glycosylation; suchmodifications may occur during polypeptide synthesis or processing orfollowing treatment with isolated modifying enzymes. Analogs can alsodiffer from the naturally-occurring polypeptides of the invention byalterations in primary sequence. These include genetic variants, bothnatural and induced (for example, resulting from random mutagenesis byirradiation or exposure to ethanemethylsulfate or by site-specificmutagenesis as described in Sambrook, Fritsch and Maniatis, MolecularCloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel etal., supra). Also included are cyclized peptides, molecules, and analogswhich contain residues other than L-amina acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., .beta. or.gamma. amino acids.

In addition to full-length polypeptides, the presently disclosed subjectmatter also provides fragments of any one of the polypeptides or peptidedomains of the presently disclosed subject matter. As used herein, theterm “a fragment” means at least 5, 10, 13, or 15 amino acids. In otherembodiments a fragment is at least 20 contiguous amino acids, at least30 contiguous amino acids, or at least 50 contiguous amino acids, and inother embodiments at least 60 to 80, 100, 200, 300 or more contiguousamino acids. Fragments of the invention can be generated by methodsknown to those skilled in the art or may result from normal proteinprocessing (e.g., removal of amino acids from the nascent polypeptidethat are not required for biological activity or removal of amino acidsby alternative mRNA splicing or alternative protein processing events).

Non-protein analogs have a chemical structure designed to mimic thefunctional activity of a protein of the invention. Such analogs areadministered according to methods of the presently disclosed subjectmatter. Such analogs may exceed the physiological activity of theoriginal polypeptide. Methods of analog design are well known in theart, and synthesis of analogs can be carried out according to suchmethods by modifying the chemical structures such that the resultantanalogs increase the anti-neoplastic activity of the originalpolypeptide when expressed in an immunoresponsive cell. These chemicalmodifications include, but are not limited to, substituting alternativeR groups and varying the degree of saturation at specific carbon atomsof a reference polypeptide. In certain embodiments, the protein analogsare relatively resistant to in vivo degradation, resulting in a moreprolonged therapeutic effect upon administration. Assays for measuringfunctional activity include, but are not limited to, those described inthe Examples below.

Pre-Leukemic Stem Cell Model

Pre-leukemic stem cells are genetically defined by the expression ofinitiating mutations including, but are not limited to, DNMT3a (Shlushet al., 2014b) and fusion protein MLLAF9.

The DNA (cytosine-5)-methyltransferase 3A (DNMT3A) is a member of theDNA methyltransferase family and one of the most frequently mutatedgenes in AML, occurring in up to 36% of cytogenetically normal AML(CN-AML) patients (Marcucci et al., 2012). A recurrent heterozygousmutation at residue Arginine 882 accounts for 40% to 60% of DNMT3Amutations (Ley et al., 2010; Yan et al., 2011). In AML cells, R882mutations always occur with retention of the wild-type allele and it wasshowed that the R882 mutant serves as a dominant-negative regulator ofwild-type DNMT3A (Russler-Germain et al., 2014).

The most common fusion protein MLLAF9 induces the inappropriateexpression of homeotic (Hox) genes, which, during normal hematopoiesis,are maintained by wild-type MLL. Studies in mice have demonstrated thatMLL-fusions can confer self-renewal activity to committed myeloidprogenitors (Cozzio et al., 2003; So et al., 2003).

Targeting Myeloid/AML Antigens with Genentically Modified T Cells

In certain non-limiting embodiments, an immunoresponsive cell (e.g., a Tcell, Tumor Infiltrating Lymphocyte, Natural Killer (NK) cell, cytotoxicT lymphocyte (CTL), Natural Killer T (NKT) cells or regulatory T cell),which comprises an antigen binding receptor (e.g., CAR or TCR) directedtoward a myeloid/AML antigen, is used to treat and/or prevent a myeloiddisorder (e.g., AML). In certain non-limiting embodiments, the antigenis selected from the group consisting of EMR2, CD33, IL10RB, PLXNC1,PIEZO1, CD300LF, CPM, ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6,ENG, SIRPB1, MRP1, ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1, CCR1, SLC22A5,TFR2, KCNN4, LILRB4, LTB4R, CD70, GYPA, FCGR1A, CD123, CLEC12A, ITGB5,PTPRJ, SLC30A1, EMC10, TNFRSF1B, CD82, ITGAX, CR1, DAGLB, SEMA4A, TLR2,P2RY13, LILRB2, EMB, CD96, LILRB3, LILRA6, LILRA2, and SLC19A1. Incertain embodiments, the antigen is selected from the group consistingof LTB4R, EMR2, CD33, MYADM, PIEZO1, SIRPB1, SLC9A1, KCNN4, ENG, ITGA5,and CD70. In certain embodiments, the antigen is selected from the groupconsisting of LTB4R, EMR2, MYADM and PIEZO1. In certain embodiments, theantigen is selected from the group consisting of CD82, TNFRSF1B, EMR2,ITGB5, CCR1, CD96, PTPRJ, CD70 and LILRB2. In certain embodiments, theantigen is selected from the group consisting of TNFRSF1B, EMR2, CCR1,CD96, CD70 and LILRB2. In certain embodiments, the antigen is selectedfrom the group consisting of EMR2, CCR1, CD70 and LILRB2.

In certain non-limiting embodiments, the antigen is a cell surface genehaving increased expression level in DNMT3a mutant cells or in MLLAF9mutant cells. In certain non-limiting embodiments, the antigen isselected from the group consisting of genes in Table 2.

In certain embodiment, a T cell is engineered to express a CAR targetinga Myeloid/AML antigen (e.g., LTB4R, EMR2, MYADM, and PIEZO1).

In certain embodiment, a T cells is engineered to comprise (e.g.,express) (a) a CAR targeting a Myeloid/AML antigen, and (b) a CCRtargeting a different Myeloid/AML antigen. The combination of thetargeted antigens can be any one selected from Table 1. In certainembodiment the combination can be any one of the following pairs oftargets: LTB4R and EMR2, LTB4R and CD33, LTB4R and ENG, LTB4R and MYADM,LTB4R and PIEZO1, LTB4R and SIRPB1, LTB4R and SLC9A1, LTB4R and ITGA5,LTB4R and CD70, LTB4R and KCNN4. EMR2 and CD33, EMR2 and ENG, EMR2 andMYADM, EMR2 and PIEZO1, EMR2 and SIRPB1, EMR2 and SLC9A1, EMR2 andITGA5, EMR2 and CD70, EMR2 and KCNN4, CD33 and ENG, CD33 and MYADM, CD33and PIEZO1, CD33 and SIRPB1, CD33 and SLC9A1, CD33 and ITGA5, CD33 andCD70, CD33 and KCNN4, ENG and MYADM, ENG and PIEZO1, ENG and SIRPB1, ENGand SLC9A1, ENG and ITGA5, ENG and CD70, ENG and KCNN4, MYADM andPIEZO1, MYADM and SIRPB1, MYADM and SLC9A1, MYADM and ITGA5, MYADM andCD70, MYADM and KCNN4, PIEZO1 and SIRPB1, PIEZO1 and SLC9A1, PIEZO1 andITGA5, PIEZO1 and CD70, PIEZO1 and KCNN4, SIRPB1 and SLC9A1, SIRPB1 andITGA5, SIRPB1 and CD70, SIRPB1 and KCNN4, SLC9A1 and ITGA5, SLC9A1 andCD70, SLC9A1 and KCNN4, ITGA5 and CD70, ITGA5 and KCNN4, CD70 and KCNN4,EMR2 and CD33, CCR1 and CLEC12A, CD70 and CD33, LILRB2 and CLEC12A, EMR2and CLEC12A, EMR2 and CD96, CCR1 and CD33, CCR1 and CD96, CD70 andCLEC12A, CD70 and CD96, LILRB2 and CD33, LILRB2 and CD96, and EMR2 andCD70. In certain embodiments, the combination is EMR2 and CD33, CCR1 andCLEC12A, CD70 and CD33, LILRB2 and CLEC12A, LTB4R1 and CD70, CD70 andEMR2, or LTB4R1 and EMR2.

In certain embodiments, the combination is selected from the groupconsisting of EMR2 and CD33, CCR1 and CLEC12A, CD70 and CD33, and LILRB2and CLEC12A.

In certain embodiment, the CAR is a second generation CAR, comprising anscFv targeting an antigen of interest, a co-stimulatory domain fromCD3ζ-chain, CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB,or CD28 signaling domain. In certain embodiment, the CAR isrecombinantly expressed (e.g., via a vector, e.g., a retroviral vector).In certain embodiment, the vector is 28z retroviral vector (see detaileddescription of 28z vector in WO 2014165707 A2, WO 2014134165 A1, and WO2016042461 A1, which are incorporated by reference in their entireties).In certain embodiment, the CCR is recombinantly expressed (e.g., via avector, e.g., a retroviral vector). In certain embodiment, the vectorcomprises an scFv targeting an antigen of interest, a CD28 transmembraneand signaling domain, fused to a 4-1 BB (aka CD137) cytosoiic signalingdomain (e.g., 28BB CCR, see detailed description in WO2014055668 A1,which is incorporated by reference in its entirety). In certainembodiment, the T cell is autologous.

TABLE 1 [CRR1 + SLC22A5] [SLC19A1 + CD300LF] [LILRB4 + CD33] [CD300LF +SLC43A3] [CRR1 + TFR2] [SLC19A1 + CPM] [LILRB4 + IL10RB] [CD300LF +MYADM] [CRR1 + KCNN4] [SLC19A1 + ITFG3] [LILRB4 + PLNXC1] [CD300LF +ICAM1] [CRR1 + LILRB4] [SLC19A1 + TTYH3] [LILRB4 + PIEZO1] [CD300LF +SLC44A1] [CRR1 + LTB4R] [SLC19A1 + ITGA4] [LILRB4 + CD300LF] [CPM +ITFG3] [CRR1 + CD70] [SLC19A1 + SLC9A1] [LILRB4 + CPM] [CPM + TTYH3][CRR1 + GYPA] [SLC19A1 + MBOAT7] [LILRB4 + ITFG3] [CPM + ITGA4] [CRR1 +FCGR1A] [SLC19A1 + CD38] [LILRB4 + TTYH3] [CPM + SLC9A1] [CRR1 +SLC19A1] [SLC19A1 + SLC6A6] [LILRB4 + ITGA4] [CPM + MBOAT7] [CRR1 +EMR2] [SLC19A1 + ENG] [LILRB4 + SLC9A1] [CPM + CD38] [CRR1 + CD33][SLC19A1 + SIRPB1] [LILRB4 + MBOAT7] [CPM + SLC6A6] [CRR1 + IL10RB][SLC19A1 + MRP1] [LILRB4 + CD38] [CPM + ENG] [CRR1 + PLNXC1] [SLC19A1 +ITGA5] [LILRB4 + SLC6A6] [CPM + SIRPB1] [CRR1 + PIEZO1] [SLC19A1 +SLC43A3] [LILRB4 + ENG] [CPM + MRP1] [CRR1 + CD300LF] [SLC19A1 + MYADM][LILRB4 + SIRPB1] [CPM + ITGA5] [CRR1 + CPM] [SLC19A1 + ICAM1] [LILRB4 +MRP1] [CPM + SLC43A3] [CRR1 + ITFG3] [SLC19A1 + SLC44A1] [LILRB4 +ITGA5] [CPM + MYADM] [CRR1 + TTYH3] [EMR2 + CD33] [LILRB4 + SLC43A3][CPM + ICAM1] [CRR1 + ITGA4] [EMR2 + IL10RB] [LILRB4 + MYADM] [CPM +SLC44A1] [CRR1 + SLC9A1] [EMR2 + PLNXC1] [LILRB4 + ICAM1] [ITFG3 +TTYH3] [CRR1 + MBOAT7] [EMR2 + PIEZO1] [LILRB4 + SLC44A1] [ITFG3 +ITGA4] [CRR1 + CD38] [EMR2 + CD300LF] [LTB4R + CD70] [ITFG3 + SLC9A1][CRR1 + SLC6A6] [EMR2 + CPM] [LTB4R + GYPA] [ITFG3 + MBOAT7] [CRR1 +ENG] [EMR2 + ITFG3] [LTB4R + FCGR1A] [ITFG3 + CD38] [CRR1 + SIRPB1][EMR2 + TTYH3] [LTB4R + SLC19A1] [ITFG3 + SLC6A6] [CRR1 + MRP1] [EMR2 +ITGA4] [LTB4R + EMR2] [ITFG3 + ENG] [CRR1 + ITGA5] [EMR2 + SLC9A1][LTB4R + CD33] [ITFG3 + SIRPB1] [CRR1 + SLC43A3] [EMR2 + MBOAT7][LTB4R + IL10RB] [ITFG3 + MRP1] [CRR1 + MYADM] [EMR2 + CD38] [LTB4R +PLNXC1] [ITFG3 + ITGA5] [CRR1 + ICAM1] [EMR2 + SLC6A6] [LTB4R + PIEZO1][ITFG3 + SLC43A3] [CRR1 + SLC44A1] [EMR2 + ENG] [LTB4R + CD300LF][ITFG3 + MYADM] [SLC22A5 + TFR2] [EMR2 + SIRPB1] [LTB4R + CPM] [ITFG3 +ICAM1] [SLC22A5 + KCNN4] [EMR2 + MRP1] [LTB4R + ITFG3] [ITFG3 + SLC44A1][SLC22A5 + LILRB4] [EMR2 + ITGA5] [LTB4R + TTYH3] [TTYH3 + ITGA4][SLC22A5 + LTB4R] [EMR2 + SLC43A3] [LTB4R + ITGA4] [TTYH3 + SLC9A1][SLC22A5 + CD70] [EMR2 + MYADM] [LTB4R + SLC9A1] [TTYH3 + MBOAT7][SLC22A5 + GYPA] [EMR2 + ICAM1] [LTB4R + MBOAT7] [TTYH3 + CD38][SLC22A5 + FCGR1A] [EMR2 + SLC44A1] [LTB4R + CD38] [TTYH3 + SLC6A6][SLC22A5 + SLC19A1] [CD33 + IL10RB] [LTB4R + SLC6A6] [TTYH3 + ENG][SLC22A5 + EMR2] [CD33 + PLNXC1] [LTB4R + ENG] [TTYH3 + SIRPB1][SLC22A5 + CD33] [CD33 + PIEZO1] [LTB4R + SIRPB1] [TTYH3 + MRP1][SLC22A5 + IL10RB] [CD33 + CD300LF] [LTB4R + MRP1] [TTYH3 + ITGA5][SLC22A5 + PLNXC1] [CD33 + CPM] [LTB4R + ITGA5] [TTYH3 + SLC43A3][SLC22A5 + PIEZO1] [CD33 + ITFG3] [LTB4R + SLC43A3] [TTYH3 + MYADM][SLC22A5 + CD300LF] [CD33 + TTYH3] [LTB4R + MYADM] [TTYH3 + ICAM1][SLC22A5 + CPM] [CD33 + ITGA4] [LTB4R + ICAM1] [TTYH3 + SLC44A1][SLC22A5 + ITFG3] [CD33 + SLC9A1] [LTB4R + SLC44A1] [ITGA4 + SLC9A1][SLC22A5 + TTYH3] [CD33 + MBOAT7] [CD70 + GYPA] [ITGA4 + MBOAT7][SLC22A5 + ITGA4] [CD33 + CD38] [CD70 + FCGR1A] [ITGA4 + CD38][SLC22A5 + SLC9A1] [CD33 + SLC6A6] [CD70 + SLC19A1] [ITGA4 + SLC6A6][SLC22A5 + MBOAT7] [CD33 + ENG] [CD70 + EMR2] [ITGA4 + ENG] [SLC22A5 +CD38] [CD33 + SIRPB1] [CD70 + CD33] [ITGA4 + SIRPB1] [SLC22A5 + SLC6A6][CD33 + MRP1] [CD70 + IL10RB] [ITGA4 + MRP1] [SLC22A5 + ENG] [CD33 +ITGA5] [CD70 + PLNXC1] [ITGA4 + ITGA5] [SLC22A5 + SIRPB1] [CD33 +SLC43A3] [CD70 + PIEZO1] [ITGA4 + SLC43A3] [SLC22A5 + MRP1] [CD33 +MYADM] [CD70 + CD300LF] [ITGA4 + MYADM] [SLC22A5 + ITGA5] [CD33 + ICAM1][CD70 + CPM] [ITGA4 + ICAM1] [SLC22A5 + SLC43A3] [CD33 + SLC44A1][CD70 + ITFG3] [ITGA4 + SLC44A1] [SLC22A5 + MYADM] [IL10RB + PLNXC1][CD70 + TTYH3] [SLC9A1 + MBOAT7] [SLC22A5 + ICAM1] [IL10RB + PIEZO1][CD70 + ITGA4] [SLC9A1 + CD38] [SLC22A5 + SLC44A1] [IL10RB + CD300LF][CD70 + SLC9A1] [SLC9A1 + SLC6A6] [TFR2 + KCNN4] [IL10RB + CPM] [CD70 +MBOAT7] [SLC9A1 + ENG] [TFR2 + LILRB4] [IL10RB + ITFG3] [CD70 + CD38][SLC9A1 + SIRPB1] [TFR2 + LTB4R] [IL10RB + TTYH3] [CD70 + SLC6A6][SLC9A1 + MRP1] [TFR2 + CD70] [IL10RB + ITGA4] [CD70 + ENG] [SLC9A1 +ITGA5] [TFR2 + GYPA] [IL10RB + SLC9A1] [CD70 + SIRPB1] [SLC9A1 +SLC43A3] [TFR2 + FCGR1A] [IL10RB + MBOAT7] [CD70 + MRP1] [SLC9A1 +MYADM] [TFR2 + SLC19A1] [IL10RB + CD38] [CD70 + ITGA5] [SLC9A1 + ICAM1][TFR2 + EMR2] [IL10RB + SLC6A6] [CD70 + SLC43A3] [SLC9A1 + SLC44A1][TFR2 + CD33] [IL10RB + ENG] [CD70 + MYADM] [MBOAT7 + CD38] [TFR2 +IL10RB] [1L10RB + SIRPB1] [CD70 + ICAM1] [MBOAT7 + SLC6A6] [TFR2 +PLNXC1] [IL10RB + MRP1] [CD70 + SLC44A1] [MBOAT7 + ENG] [TFR2 + PIEZO1][IL10RB + ITGA5] [GYPA + FCGR1A] [MBOAT7 + SIRPB1] [TFR2 + CD300LF][IL10RB + SLC43A3] [GYPA + SLC19A1] [MBOAT7 + MRP1] [TFR2 + CPM][IL10RB + MYADM] [GYPA + EMR2] [MBOAT7 + ITGA5] [TFR2 + ITFG3] [IL10RB +ICAM1] [GYPA + CD33] [MBOAT7 + SLC43A3] [TFR2 + TTYH3] [IL10RB +SLC44A1] [GYPA + IL10RB] [MBOAT7 + MYADM] [TFR2 + ITGA4] [PLNXC1 +PIEZO1] [GYPA + PLNXC1] [MBOAT7 + ICAM1] [TFR2 + SLC9A1] [PLNXC1 +CD300LF] [GYPA + PIEZO1] [MBOAT7 + SLC44A1] [TFR2 + MBOAT7] [PLNXC1 +CPM] [GYPA + CD300LF] [CD38 + SLC6A6] [TFR2 + CD38] [PLNXC1 + ITFG3][GYPA + CPM] [CD38 + ENG] [TFR2 + SLC6A6] [PLNXC1 + TTYH3] [GYPA +ITFG3] [CD38 + SIRPB1] [TFR2 + ENG] [PLNXC1 + ITGA4] [GYPA + TTYH3][CD38 + MRP1] [TFR2 + SIRPB1] [PLNXC1 + SLC9A1] [GYPA + ITGA4] [CD38 +ITGA5] [TFR2 + MRP1] [PLNXC1 + MBOAT7] [GYPA + SLC9A1] [CD38 + SLC43A3][TFR2 + ITGA5] [PLNXC1 + CD38] [GYPA + MBOAT7] [CD38 + MYADM] [TFR2 +SLC43A3] [PLNXC1 + SLC6A6] [GYPA + CD38] [CD38 + ICAM1] [TFR2 + MYADM][PLNXC1 + ENG] [GYPA + SLC6A6] [CD38 + SLC44A1] [TFR2 + ICAM1] [PLNXC1 +SIRPB1] [GYPA + ENG] [SLC6A6 + ENG] [TFR2 + SLC44A1] [PLNXC1 + MRP1][GYPA + SIRPB1] [SLC6A6 + SIRPB1] [KCNN4 + LILRB4] [PLNXC1 + ITGA5][GYPA + MRP1] [SLC6A6 + MRP1] [KCNN4 + LTB4R] [PLNXC1 + SLC43A3] [GYPA +ITGA5] [SLC6A6 + ITGA5] [KCNN4 + CD70] [PLNXC1 + MYADM] [GYPA + SLC43A3][SLC6A6 + SLC43A3] [KCNN4 + GYPA] [PLNXC1 + ICAM1] [GYPA + MYADM][SLC6A6 + MYADM] [KCNN4 + FCGR1A] [PLNXC1 + SLC44A1] [GYPA + ICAM1][SLC6A6 + ICAM1] [KCNN4 + SLC19A1] [PIEZO1 + CD300LF] [GYPA + SLC44A1][SLC6A6 + SLC44A1] [KCNN4 + EMR2] [PIEZO1 + CPM] [FCGR1A + SLC19A1][ENG + SIRPB1] [KCNN4 + CD33] [PIEZO1 + ITFG3] [FCGR1A + EMR2] [ENG +MRP1] [KCNN4 + IL10RB] [PIEZO1 + TTYH3] [FCGR1A + CD33] [ENG + ITGA5][KCNN4 + PLNXC1] [PIEZO1 + ITGA4] [FCGR1A + IL10RB] [ENG + SLC43A3][KCNN4 + PIEZO1] [PIEZO1 + SLC9A1] [FCGR1A + PLNXC1] [ENG + MYADM][KCNN4 + CD300LF] [PIEZO1 + MBOAT7] [FCGR1A + PIEZO1] [ENG + ICAM1][KCNN4 + CPM] [PIEZO1 + CD38] [FCGR1A + CD300LF] [ENG + SLC44A1][KCNN4 + ITFG3] [PIEZO1 + SLC6A6] [FCGR1A + CPM] [SIRPB1 + MRP1][KCNN4 + TTYH3] [PIEZO1 + ENG] [FCGR1A + ITFG3] [SIRPB1 + ITGA5][KCNN4 + ITGA4] [PIEZO1 + SIRPB1] [FCGR1A + TTYH3] [SIRPB1 + SLC43A3][KCNN4 + SLC9A1] [PIEZO1 + MRP1] [FCGR1A + ITGA4] [SIRPB1 + MYADM][KCNN4 + MBOAT7] [PIEZO1 + ITGA5] [FCGR1A + SLC9A1] [SIRPB1 + ICAM1][KCNN4 + CD38] [PIEZO1 + SLC43A3] [FCGR1A + MBOAT7] [SIRPB1 + SLC44A1][KCNN4 + SLC6A6] [PIEZO1 + MYADM] [FCGR1A + CD38] [MRP1 + ITGA5][KCNN4 + ENG] [PIEZO1 + ICAM1] [FCGR1A + SLC6A6] [MRP1 + SLC43A3][KCNN4 + SIRPB1] [PIEZO1 + SLC44A1] [FCGR1A + ENG] [MRP1 + MYADM][KCNN4 + MRP1] [CD300LF + CPM] [FCGR1A + SIRPB1] [MRP1 + ICAM1] [KCNN4 +ITGA5] [CD300LF + ITFG3] [FCGR1A + MRP1] [MRP1 + SLC44A1] [KCNN4 +SLC43A3] [CD300LF + TTYH3] [FCGR1A + ITGA5] [ITGA5 + SLC43A3] [KCNN4 +MYADM] [CD300LF + ITGA4] [FCGR1A + SLC43A3] [ITGA5 + MYADM] [KCNN4 +ICAM1] [CD300LF + SLC9A1] [FCGR1A + MYADM] [ITGA5 + ICAM1] [KCNN4 +SLC44A1] [CD300LF + MBOAT7] [FCGR1A + ICAM1] [ITGA5 + SLC44A1] [LILRB4 +LTB4R] [CD300LF + CD38] [FCGR1A + SLC44A1] [SLC43A3 + MYADM] [LILRB4 +CD70] [CD300LF + SLC6A6] [SLC19A1 + EMR2] [SLC43A3 + ICAM1] [LILRB4 +GYPA] [CD300LF + ENG] [SLC19A1 + CD33] [SLC43A3 + SLC44A1] [LILRB4 +FCGR1A] [CD300LF + SIRPB1] [SLC19A1 + IL10RB] [MYADM + ICAM1] [LILRB4 +SLC19A1] [CD300LF + MRP1] [SLC19A1 + PLNXC1] [MYADM + SLC44A1] [LILRB4 +EMR2] [CD300LF + ITGA5] [SLC19A1 + PIEZO1] [ICAM1 + SLC44A1]

TABLE 2 DNMT3a Mutant MLLAF9 mutant TMEM40 ABCG2 CEACAM6 GNAZ ANO9MANSC1 SLC6A16 GAS2 ELOVL6 PPP2R5B ASIC3 LEPR TEX29 B3GNT4 SUN3 FKBP1BTMEM59L HOOK1 KCNJ5 SLC25A36 CCDC155 CAPN3 FRMD5 TMEM27 TNFRSF14 COL15A1GABRB2 SPAG17 ZDHHC11 EPHA4 MMP25 ITGA8 CDH13 NGFR PEAR1 AQP2 CLEC1AASPRV1 KCNK13 OTOA LOXL4 KIF26B LRRN2 TRIM55 HTR2A RHBDL3 KIF19 SLC44A3HEPHL1 LPAR2 ILDR1 TSPEAR CNIH2 CYP4F11 TAS1R3 FLRT1 SLC8A3 MBOAT1RNF183 GPR153 MT-ND1 RDH16 SLCO2B1 DARC CADM3 SCIN SH3PXD2A C3orf35SCN2A BEST4 GDPD3 IL23R STON2 TMPRSS5 ALS2 ACKR6 SEC31B GNA14 LRRTM2AGER TMEFF2 STC1 ADAMTS13 EXTL3 SLC16A6 IL20RB PDE3A CDHR1 WNT4 MFAP3LMYADML2 LRRC37A3 SLC34A3 PNPLA3 SCNN1D TACSTD2 PSD2 TMEM89 ITGB8SLC25A41 EXOC3L4 LAX1 SUSD2 ATP6V0A4 SLC45A3 KCND1 CHST3 SYNC HILPDANPAS2 PLXNA4 TMEM145 IGFBP3 ADORA3 DFNB31 ADRA1D SIGLEC11 PPFIA4 RNF173RYR2 NLGN3 LRRC8E FAM186B DGKI KCNV2 COLEC12 SCN11A CX3CR1Genomice Integration into Immunoresponsive Cell

In certain embodiments, an antigen recognizing receptor (e.g., a CAR ora TCR) can be integrated into a selected locus of the genome of animmunoresponsive cell. Any targeted genome editing methods can be usedto integrate the antigen recognizing receptor (e.g., CAR or TCR) inselected loci of the genome of an immunoresponsive cell. In certainembodiments, the expression of the antigen recognizing receptor (e.g.,CAR or TCR) is driven by an endogenous promoter/enhancer within or nearthe locus. In certain embodiments, the expression of the antigenrecognizing receptor (e.g., CAR or TCR) is driven by an exogenouspromoter integrated into the locus. The locus where the antigenrecognizing receptor (e.g., CAR or TCR) is integrated is selected basedon the expression level of the genes within the locus, and timing of thegene expression of the genes within the locus. The expression level andtiming can vary under different stages of cell differentiation andmitogen/cytokine microenvironment, which are among the factors to beconsidered when making the selection.

In certain embodiments, the CRISPR system is used to integrate theantigen recognizing receptor (e.g., CAR or TCR) in selected loci of thegenome of an immunoresponsive cell. Clustered regularly-interspacedshort palindromic repeats (CRISPR) system is a genome editing tooldiscovered in prokaryotic cells. When utilized for genome editing, thesystem includes Cas9 (a protein able to modify DNA utilizing crRNA asits guide), CRISPR RNA (crRNA, contains the RNA used by Cas9 to guide itto the correct section of host DNA along with a region that binds totracrRNA (generally in a hairpin loop form) forming an active complexwith Cas9), trans-activating crRNA (tracrRNA, binds to crRNA and formsan active complex with Cas9), and an optional section of DNA repairtemplate (DNA that guides the cellular repair process allowing insertionof a specific DNA sequence). CRISPR/Cas9 often employs a plasmid totransfect the target cells. The crRNA needs to be designed for eachapplication as this is the sequence that Cas9 uses to identify anddirectly bind to the target DNA in a cell. The repair template carryingCAR expression cassette need also be designed for each application, asit must overlap with the sequences on either side of the cut and codefor the insertion sequence. Multiple crRNA's and the tracrRNA can bepackaged together to form a single-guide RNA (sgRNA). This sgRNA can bejoined together with the Cas9 gene and made into a plasmid in order tobe transfected into cells. Methods of using the CRISPR system aredescribed, for example, in WO 2014093661 A2, WO 2015123339 A1 and WO2015089354 A1, which are incorporated by reference in their entireties.

In certain embodiments, zinc-finger nucleases are used to integrate theantigen recognizing receptor (e.g., CAR or TCR) in selected loci of thegenome of an immunoresponsive cell. A zinc-finger nuclease (ZFN) is anartificial restriction enzyme, which is generated by combining a zincfinger DNA-binding domain with a DNA-cleavage domain. A zinc fingerdomain can be engineered to target specific DNA sequences which allows azinc-finger nuclease to target desired sequences within genomes. TheDNA-binding domains of individual ZFNs typically contain a plurality ofindividual zinc finger repeats and can each recognize a plurality ofbasepairs. The most common method to generate new zinc-finger domain isto combine smaller zinc-finger “modules” of known specificity. The mostcommon cleavage domain in ZFNs is the non-specific cleavage domain fromthe type IIs restriction endonuclease FokI. Using the endogenoushomologous recombination (HR) machinery and a homologous DNA templatecarrying CAR expression cassette, ZFNs can be used to insert the CARexpression cassette into genome. When the targeted sequence is cleavedby ZFNs, the HR machinery searches for homology between the damagedchromosome and the homologous DNA template, and then copies the sequenceof the template between the two broken ends of the chromosome, wherebythe homologous DNA template is integrated into the genome. Methods ofusing the ZFN system are described, for example, in WO 2009146179 A1, WO2008060510 A2 and CN 102174576 A, which are incorporated by reference intheir entireties.

In certain embodiments, the TALEN system is used to integrate theantigen recognizing receptor (e.g., CAR or TCR) in selected loci of thegenome of an immunoresponsive cell. Transcription activator-likeeffector nucleases (TALEN) are restriction enzymes that can beengineered to cut specific sequences of DNA. TALEN system operates onalmost the same principle as ZFNs. They are generated by combining atranscription activator-like effectors DNA-binding domain with a DNAcleavage domain. Transcription activator-like effectors (TALEs) arecomposed of 33-34 amino acid repeating motifs with two variablepositions that have a strong recognition for specific nucleotides. Byassembling arrays of these TALEs, the TALE DNA-binding domain can beengineered to bind desired DNA sequence, and thereby guide the nucleaseto cut at specific locations in genome. Methods of using the TALENsystem are described, for example, in WO 2014134412 A1, WO 2013163628 A2and WO 2014040370 A1, which are incorporated by reference in theirentireties.

Methods for delivering the genome editing agents can vary depending onthe need. In certain embodiments, the components of a selected genomeediting method are delivered as DNA constructs in one or more plasmids.In certain embodiments, the components are delivered via viral vectors.Common delivery methods include but is not limited to, electroporation,microinjection, gene gun, impalefection, hydrostatic pressure,continuous infusion, sonication, magnetofection, adeno-associatedviruses, envelope protein pseudotyping of viral vectors,replication-competent vectors cis and trans-acting elements, herpessimplex virus, and chemical vehicles (e.g., oligonucleotides,lipoplexes, polymersomes, polyplexes, dendrimers, inorganicNanoparticles, and cell-penetrating peptides).

Modification can be made anywhere within the selected locus, or anywherethat can influence gene expression of the integrated antigen recognizingreceptor (e.g., CAR or TCR). In certain embodiments, the modification isintroduced upstream of the transcriptional start site of the integratedantigen recognizing receptor (e.g., CAR or TCR). In certain embodiments,the modification is introduced between the transcriptional start siteand the protein coding region of the integrated antigen recognizingreceptor (e.g., CAR or TCR). In certain embodiments, the modification isintroduced downstream of the protein coding region of the integratedantigen recognizing receptor (e.g., CAR or TCR).

Administration

Compositions comprising genetically modified immunoresponsive cells ofthe invention (e.g., T cells, NK cells, CTL cells, or their progenitors)can be provided systemically or directly to a subject for the treatmentof a myeloid disorder. In certain embodiments, the presently disclosedcells are directly injected into an organ of interest (e.g., an organaffected by a myeloid disorder). Alternatively, compositions comprisinggenetically modified immunoresponsive cells are provided indirectly tothe organ of interest, for example, by administration into thecirculatory system (e.g., the tumor vasculature). Expansion anddifferentiation agents can be provided prior to, during or afteradministration of the cells to increase production of T cells, NK cells,or CTL cells in vitro or in vivo.

The modified cells can be administered in any physiologically acceptablevehicle, normally intravascularly, although they may also be introducedinto bone or other convenient site where the cells may find anappropriate site for regeneration and differentiation (e.g., thymus).Usually, at least 1×10⁵ cells will be administered, eventually reaching1×10¹⁰ or more. Genetically modified immunoresponsive cells of theinvention can comprise a purified population of cells. Those skilled inthe art can readily determine the percentage of genetically modifiedimmunoresponsive cells in a population using various well-known methods,such as fluorescence activated cell sorting (FACS). Preferable ranges ofpurity in populations comprising genetically modified immunoresponsivecells are about 50 to about 55%, about 55 to about 60%, and about 65 toabout 70%. More preferably the purity is about 70 to about 75%, about 75to about 80%, about 80 to about 85%; and still more preferably thepurity is about 85 to about 90%, about 90 to about 95%, and about 95 toabout 100%. Dosages can be readily adjusted by those skilled in the art(e.g., a decrease in purity may require an increase in dosage). Thecells can be introduced by injection, catheter, or the like. If desired,factors can also be included, including, but not limited to,interleukins, e.g. IL-2, IL-3, IL-6, IL-11, IL7, IL12, IL1S, IL21, aswell as the other interleukins, the colony stimulating factors, such asG-, M- and GM-CSF, interferons, e.g. .gamma.-interferon anderythropoietin.

In certain embodiments, the compositions are pharmaceutical compositionscomprising genetically modified immunoresponsive cells or theirprogenitors and a pharmaceutically acceptable carrier. Administrationcan be autologous or heterologous. For example, immunoresponsive cells,or progenitors can be obtained from one subject, and administered to thesame subject or a different, compatible subject. Peripheral bloodderived immunoresponsive cells of the invention or their progeny (e.g.,in vivo, ex vivo or in vitro derived) can be administered via localizedinjection, including catheter administration, systemic injection,localized injection, intravenous injection, or parenteraladministration. When administering a presently disclosed therapeuticcomposition (e.g., a pharmaceutical composition containing a geneticallymodified immunoresponsive cell), it will generally be formulated in aunit dosage inj ectable form (solution, suspension, emulsion).

Formulations

Presently disclosed compositions comprising genetically modifiedimmunoresponsive cells can be conveniently provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may be buffered to aselected pH. Liquid preparations are normally easier to prepare thangels, other viscous compositions, and solid compositions. Additionally,liquid compositions are somewhat more convenient to administer,especially by injection. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with specific tissues. Liquid or viscous compositionscan comprise carriers, which can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating thegenetically modified immunoresponsive cells utilized in practicing thepresent invention in the required amount of the appropriate solvent withvarious amounts of the other ingredients, as desired. Such compositionsmay be in admixture with a suitable carrier, diluent, or excipient suchas sterile water, physiological saline, glucose, dextrose, or the like.The compositions can also be lyophilized. The compositions can containauxiliary substances such as wetting, dispersing, or emulsifying agents(e.g., methylcellulose), pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the presently disclosedsubject matter, however, any vehicle, diluent, or additive used wouldhave to be compatible with the genetically modified immunoresponsivecells or their progenitors.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thepresently disclosed compositions may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The concentration ofthe thickener can depend upon the agent selected. The important point isto use an amount that will achieve the selected viscosity. Obviously,the choice of suitable carriers and other additives will depend on theexact route of administration and the nature of the particular dosageform, e.g., liquid dosage form (e.g., whether the composition is to beformulated into a solution, a suspension, gel or another liquid form,such as a time release form or liquid-filled form).

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert and will notaffect the viability or efficacy of the genetically modifiedimmunoresponsive cells as described in the presently disclosed subjectmatter. This will present no problem to those skilled in chemical andpharmaceutical principles, or problems can be readily avoided byreference to standard texts or by simple experiments (not involvingundue experimentation), from this disclosure and the documents citedherein.

One consideration concerning the therapeutic use of the presentlydisclosed immunoresponsive cells is the quantity of cells necessary toachieve an optimal effect. The quantity of cells to be administered willvary for the subject being treated. In certain embodiments, between 10⁴to 10¹⁰ between 10⁵ to 10⁹, or between 10⁶ and 10⁸ genetically presentlydisclosed cells are administered to a human subject. More effectivecells may be administered in even smaller numbers. In some embodiments,at least about 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, and 5×10⁸ presently disclosedcells are administered to a human subject. The precise determination ofwhat would be considered an effective dose may be based on factorsindividual to each subject, including their size, age, sex, weight, andcondition of the particular subject. Dosages can be readily ascertainedby those skilled in the art from this disclosure and the knowledge inthe art.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active cell(s) and/or agent(s)) are present in an amountof 0.001 to 50% (weight) solution in phosphate buffered saline, and theactive ingredient is present in the order of micrograms to milligrams,such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1wt %, still more preferably about 0.0001 to about 0.05 wt % or about0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, andstill more preferably about 0.05 to about 5 wt %. Of course, for anycomposition to be administered to an animal or human, and for anyparticular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD50 in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

Methods of Treatment

Provided herein are methods for treating a myeloid disorder in asubject. Also contemplated herein are methods for treating a pathogeninfection or other infectious disease in a subject, such as animmunocompromised human subject. The methods comprise administering thepresently disclosed cells in an amount effective to achieve the desiredeffect, be it palliation of an existing condition or prevention ofrecurrence. For treatment, the amount administered is an amounteffective in producing the desired effect. An effective amount can beprovided in one or a series of administrations. An effective amount canbe provided in a bolus or by continuous perfusion.

An “effective amount” (or, “therapeutically effective amount”) is anamount sufficient to effect a beneficial or desired clinical result upontreatment. An effective amount can be administered to a subject in oneor more doses. In terms of treatment, an effective amount is an amountthat is sufficient to palliate, ameliorate, stabilize, reverse or slowthe progression of the disease, or otherwise reduce the pathologicalconsequences of the disease. The effective amount is generallydetermined by the physician on a case-by-case basis and is within theskill of one in the art. Several factors are typically taken intoaccount when determining an appropriate dosage to achieve an effectiveamount. These factors include age, sex and weight of the subject, thecondition being treated, the severity of the condition and the form andeffective concentration of the immunoresponsive cells administered.

For adoptive immunotherapy using antigen-specific T cells, cell doses inthe range of about 10⁶-10¹⁰ (e.g., about 10⁹) are typically infused.Upon administration of the presently disclosed cells into the host andsubsequent differentiation, T cells are induced that are specificallydirected against the specific antigen. “Induction” of T cells caninclude inactivation of antigen-specific T cells such as by deletion oranergy. Inactivation is particularly useful to establish or reestablishtolerance such as in autoimmune disorders. The modified cells can beadministered by any method known in the art including, but not limitedto, intravenous, subcutaneous, intranodal, intratumoral, intrathecal,intrapleural, intraperitoneal and directly to the thymus.

Therapeutic Methods

The presently disclosed subject matter provides methods for increasingan immune response in a subject in need thereof. The presently disclosedsubject matter provides methods for treating and/or preventing a myeloiddisorder in a subject. Suitable human subjects for therapy typicallycomprise two treatment groups that can be distinguished by clinicalcriteria. Subjects with “advanced disease” or “high tumor burden” arethose who bear a clinically measurable tumor. A clinically measurabletumor is one that can be detected on the basis of tumor mass (e.g.,based on percentage of leukemic cells, by palpation, CAT scan, sonogram,mammogram or X-ray; positive biochemical or histopathologic markers ontheir own are insufficient to identify this population). Apharmaceutical composition embodied in this invention is administered tothese subjects to elicit an anti-tumor response, with the objective ofpalliating their condition. Ideally, reduction in tumor mass occurs as aresult, but any clinical improvement constitutes a benefit. Clinicalimprovement includes decreased risk or rate of progression or reductionin pathological consequences of the tumor.

A second group of suitable subjects is known in the art as the “adjuvantgroup.” These are individuals who have had a history of a myeloiddisorder, but have been responsive to another mode of therapy. The priortherapy can have included, but is not restricted to, surgical resection,radiotherapy, and traditional chemotherapy. As a result, theseindividuals have no clinically measurable tumor. However, they aresuspected of being at risk for progression of the disease, either nearthe original tumor site, or by metastases. This group can be furthersubdivided into high-risk and low-risk individuals. The subdivision ismade on the basis of features observed before or after the initialtreatment. These features are known in the clinical arts, and aresuitably defined for each different myeloid disorder. Features typicalof high-risk subgroups are those in which the tumor has invadedneighboring tissues, or who show involvement of lymph nodes.

Another group have a genetic predisposition to a myeloid disorder buthave not yet evidenced clinical signs of the myeloid disorder. Forinstance, women testing positive for a genetic mutation associated withAML, but still of childbearing age, can wish to receive one or more ofthe immunoresponsive cells described herein in treatmentprophylactically to prevent the occurrence of AML until it is suitableto perform preventive surgery.

The subjects can have an advanced form of disease, in which case thetreatment objective can include mitigation or reversal of diseaseprogression, and/or amelioration of side effects. The subjects can havea history of the condition, for which they have already been treated, inwhich case the therapeutic objective will typically include a decreaseor delay in the risk of recurrence.

Accordingly, the presently disclosed subject matter provides a method oftreating and/or preventing a myeloid disorder in a subject, the methodcomprising administering an effective amount of the presently disclosedimmunoresponsive cells.

As a consequence of surface expression of a receptor that binds to amyeloid disorder associated antigen and activates the immunoresponsivecell that enhances the anti-myeloid cell effect of the immunoresponsivecell, adoptively transferred human T or NK cells are endowed withaugmented and selective cytolytic activity at the treatment site.Furthermore, subsequent to their localization to treatment site andtheir proliferation, the T cells turn the site into a highly conductiveenvironment for a wide range of immune cells involved in thephysiological immune response (tumor infiltrating lymphocytes, NK-,NKT-cells, dendritic cells, and macrophages).

Kits

The invention provides kits for the treatment and/or prevention of amyeloid disorder. In certain embodiments, the kit includes a therapeuticor prophylactic composition comprising an effective amount of thepresently disclosed immunoresponsive cells. In some embodiments, the kitcomprises a sterile container; such containers can be boxes, ampules,bottles, vials, tubes, bags, pouches, blister-packs, or other suitablecontainer forms known in the art. Such containers can be made ofplastic, glass, laminated paper, metal foil, or other materials suitablefor holding medicaments. In certain embodiments, the kit includes anisolated nucleic acid encoding an antigen recognizing receptor (e.g., aCAR or a TCR) directed toward an antigen of interest in expessible (andsecretable) form, which may optionally be comprised in the same ordifferent vectors.

If desired, the immunoresponsive cell and/or nucleic acid is providedtogether with instructions for administering the cell or nucleic acid toa subject having or at risk of developing a myeloid disorder. Theinstructions will generally include information about the use of thecomposition for the treatment and/or prevention of myeloid disorder. Incertain embodiments, the instructions include at least one of thefollowing: description of the therapeutic agent; dosage schedule andadministration for treatment or prevention of a myeloid disorder or asymptom thereof; precautions; warnings; indications;counter-indications; overdosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions maybe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container.

EXAMPLES

The practice of the present disclosure employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions, and assay, screening, and therapeuticmethods of the invention, and are not intended to limit the scope ofwhat the inventors regard as their invention.

Example 1—Integrated Analysis of the AML Cell Surfaceome Identifies CARTargets Summary

Adoptive T cell therapies using chimeric antigen receptors (CARs) toredirect the specificity and function of T lymphocytes have demonstratedefficacy in patients with lymphoid malignancies, in particular acutelymphoblastic leukemia (ALL) (Sadelain, 2015). This therapeutic modalityinduces complete remissions in subjects with CD19⁺ malignancies for whomchemotherapies have led to drug resistance and tumor progression.“Cancer immunotherapy”, including CAR therapy, was proclaimed ascientific breakthrough in 2013 (Couzin-Frankel, 2013). The success ofCD19 CAR therapy bodes well for tackling all hematological malignancies,including Acute Myeloid Leukemia (AML), which affects over one quartermillion adults annually worldwide. However, the development of CARtherapy for AML is hampered by the lack of suitable targets.

The search for suitable CAR targets has been limited so far tocomparisons of antigen expression levels between cancer cells and normalcounterparts, without comprehensively considering antigen expression innormal organs/tissues across the whole body. Such searches have mostlyrelied on the analysis of the transcriptome, under the assumption thatthere exists a direct correspondence between mRNA transcripts andgenerated protein expressions. Alternatively, advanced proteomictechnologies combined to enrichment strategies to identify plasma cellmembrane proteins offer direct measurements of the surface proteome.Recent studies have however shown that the correlation between mRNA andprotein expressions is complex and sometimes unreliable due to variousfactors including different half lives and post transcription machinery.Therefore, an integrated and comprehensive analysis of bothtranscriptomic and proteomic data is best positioned to capture usefulinformation that may not be deciphered from individual or limitedanalysis of mRNA or protein expression (Haider and Pal, 2013). Moreover,defined criteria of a suitable CAR target have never been set andbesides the CD19 paradigm. There is thus an unmet need to defineanalytical tools to select proper candidate targets for CAR therapy.

A multi-tiered approach was developed, integrating transcriptomic andproteomic data from several malignant and normal cell subsets in searchfor CAR targets. Starting from multiple annotation data sources,multi-step ranking criteria was defined to identify suitable CARtargets. This allowed to annotate, filter and validate thousands ofsurface potential targets, which were found overexpressed in AML cellscompared to normal controls. Eleven suitable candidate antigens wereidentified that can be targeted by single and/or combinatorial CAR Tcell strategies for patients with AML. These do not overlap with thefour molecules that have been pursued to date as CAR targets: Lewis(Le)-Y(Ritchie et al., 2013); CD123 (Al-Hussaini et al., 2016; Gill etal., 2014; Jordan et al., 2000; Yi Luo, 2015); CD33 (Kenderian et al.,2015; Pizzitola et al., 2014) and folate receptor-β (Lynn et al., 2016;Lynn et al., 2015). None of these 4 meet stringent efficacy and safetycriteria for an ideal CAR target or represent the equivalent of CD19 forALL. Furthermore, genetically-defined models of leukemic stem cells aregenerated, which were used to further validate the candidates and pairsof candidates and also to identify mutation-specific surface signatures,which may be used to design patient-tailored CAR strategies, based onthe genetic mutational profile.

Generating a Comprehensive Annotated Dataset of Surface AML Targets toIdentify Suitable CAR Targets

In order to generate a comprehensive dataset of surface AML targets,from which suitable CAR targets could be selected, previously reportedmarkers (474 proteins) were first collected, and additionalsurface-specific proteomic studies were performed (3675 proteins) (FIG.1A).

Reported markers include CD44 (Jin et al., 2006), TIM-3 (Kikushige etal., 2010), CD123 (Jordan et al., 2000), CD47 (Majeti et al., 2009),CD32 and CD25 (Saito et al., 2010), CLL-1 (Bakker et al., 2004), CD96(Hosen et al., 2007), CD33 (Taussig et al., 2005) for instance, and alsothe results of surface-specific proteomic studies in five human myeloidleukemia (NB4, HL60, THP1, PLB985, K562) cell lines and normal humangranulocytes (Strassberger et al., 2014). Also performed was cellsurface biotinylation of additional four (Kasumi, Monomac, Molm13,09AML, THP1, K562) human AML cell lines, which was used formass-spectrometric analysis. These eight cell lines bear differentgenetic background and therefore including additional lines expanded thecohort of potential candidate targets given the complex heterogenicityof AML. For instance, THP1 line bears MLL-AF9 translocation, deletion ofp16, p53, UTX and rearrangement of RB1; Kasumi line bears AML1-ETOtranslocation; Molm13 FLT3-ITD; NB4 and HL60 PML-RAR.

The expression of each candidate was annotated through multiple datasources, including the Human Protein Atlas (HPA)(Uhlen et al., 2015),the Human Proteome Map (HPM)(Kim et al., 2014) and the ProteomicsDatabase (PD)(Wilhelm et al., 2014), which provide information onprotein expression in several (>60) normal tissues/organs, includingliver, gallbladder, pancreas, stomach, duodenum, colon, rectum, testis,epididymis, prostate, breast, vagina, uterus, ovary, skin, skeletal andsmooth muscle, cerebral cortex, hippocampus, lateral ventricle,cerebellum, thyroid, bronchus, lung, heart, retina, vitreous humor, bonemarrow, lymphocytes, lymph nodes, tonsil, synovial fluid, bile, saliva,through different means such as antibody-based immunohistochemistry(HPA) and protein mass spectrometry (HPM and PD) (FIG. 1A). Byconverting and integrating these data, the expression profile of eachAML target was defined in any of those normal organ systems and tissuetypes that signify different vital and non-vital organ structures andfunctions. Through the subcellular localization database (akaCompartments), the proteins localized at the cell membrane was annotated(FIG. 1A).

HPA also provided mRNA data enabling calculation of correlation betweenprotein and mRNA expression of each candidate in any normal tissue. themRNA expression of each candidate was also studied in multiple normalhematopoietic cells, such as HSCs, myeloid progenitors, monocytes etcand primary AML patient samples bearing specific chromosomalabnormalities such as t(15; 17), t(8; 21), t(11q23)/MLL, inv(16)/t(16;16) etc (Bagger et al., 2016) (FIG. 1A). Also, upon purchasing specificantibodies of a subset of 32 selected antigens, additional informationof their expression was obtained in multiple primary cell subsets suchas healthy CAR+ T cells or oncogene-expressing CD34+ cells byflow-cytometry (FIG. 1A). All these information contributed ingenerating a comprehensive annotated dataset of AML surface targets,which is not-previously reported (FIG. 1A).

The Algorithm to Identify Suitable CAR Targets in AML

Described here is the algorithm and the criteria applied to theannotated dataset of potential AML candidates, described above inFIG. 1. This multi-step approach enabled identification of a limitednumber of suitable CAR targets, starting from a much larger number.

Starting from a dataset of 3,887 molecules, priority was first given toantigens with redundant protein expression data in at least 2 out the 3databases, which provide information on the antigen expression in normaltissues and organs (HPA, HPM and PD). This assured high level ofconfidence in the analysis. Additionally, antigens with a membraneassociated sub-localization were prioritized. This first step is termed“Quality control”, which resulted in 1,694 candidates (FIG. 1B—step#1).

Secondly, the antigens with less chance of expression by normal tissueswas selected. three selection criteriawhich would prefer the candidateswith were defined: a) low average expression in 64 normal tissues/organs(using 1 as cut-off value) b) no high expression in any tissue (using ascale 0 to 3 where 0,1,2 and 3 indicate zero, low, medium and highexpression respectively) c) low mRNA expression in HSCs compared toprimary AML samples. This was called the second step “Low expression innormal tissues” resulting in 215 candidates (FIG. 1B—step#2).

The top lowest overall expressors were selected (10) based on mean value(in increasing order): CCR1, SLC22A5, TFR2, KCNN4, LILRB4, LTB4R, CD70,GYPA, FCGR1A and SLC19A1. Also selected are the candidates that arecommonly expressed between the group of reported molecules and theresults of the proteomics studies in additional AML cell lines (FIG.1A—blue boxes). This assured broad distribution of candidate expressionacross multiple AML models, thus extended application in targeting thosecandidates. They are 29 in total and the molecules are listed below, notincluding the 10 lowest overall expressors: EMR2, CD33, IL10RB, PLXNC1,PIEZO1, CD300LF, CPM, ITFG3, TTYH3, ITGA4, SLC9A1, MBOAT7, CD38, SLC6A6,ENG, SIRPB1, MRP1, ITGA5, SLC43A3, MYADM, ICAM1, SLC44A1. This thirdstep was termed “Rank selection”, resulting in 32 proteins (FIG.1B—step#3 and FIG. 1C).

Upon purchasing specific antibodies, the expression of these selectedcandidates were systematically defined in primary CD34⁺, CD34⁺CD38⁻ HSCswhich were purified from cord blood, and in five AML cell lines byflow-cytometry. This fourth step was termed “Expression byflow-cytometry”, which selected the candidates with <5% expression innormal CD34+ and CD34+CD38− HSCs and >75% expression in 4/5 AML cells(FIG. 1B—step#4). This resulted in 11 top candidates that can betargeted by CAR T cells safely and efficiently: LTB4R, EMR2, CD33,MYADM, PIEZO1, SIRPB1, SLC9A1, KCNN4, ENG, ITGA5, CD70 (FIG. 1D).

A further step was taken to consider T cells mediate CAR therapy, theexpression of each of those 11 molecules were defined in healthyPHA-stimulated CD19+CAR T cells before and after stimulation on CD19⁺3T3cells, mimicking therapeutic cells. the candidates with <5% expressionin T cells: LTB4R, EMR2, MYADM and PIEZO1 were selected (FIG.1B—step#5). This fifth step was termed “Single CAR selection.” TargetingAML with any of those can result in efficient and safe CAR T celltherapies. The 11 top targets were paired for non-overlapping expressionin normal organs/tissues and 55 pairs of antigens were obtained from thestep termed “Combinatorial CAR selection” (FIG. 1B—step#5′; FIG. 3A).

-   -   [LTB4R+EMR2]; [LTB4R+CD33]; [LTB4R+ENG]; [LTB4R+MYADM];        [LTB4R+PIEZO1]; [LTB4R+SIRPB1];    -   [LTB4R+SLC9A1]; [LTB4R+ITGA5]; [LTB4R+CD70]; [LTB4R+KCNN4];    -   [EMR2+CD33]; [EMR2+ENG]; [EMR2+MYADM]; [EMR2+PIEZO1];        [EMR2+SIRPB1]; [EMR2+SLC9A1]; [EMR2+ITGA5]; [EMR2+CD70];        [EMR2+KCNN4];    -   [CD33+ENG]; [CD33+MYADM]; [CD33+PIEZO1]; [CD33+SIRPB1];        [CD33+SLC9A1]; [CD33+ITGA5]; [CD33+CD70]; [CD33+KCNN4];    -   [ENG+MYADM]; [ENG+PIEZO1]; [ENG+SIRPB1]; [ENG+SLC9A1];        [ENG+ITGA5]; [ENG+CD70]; [ENG+KCNN4];    -   [MYADM+PIEZO1]; [MYADM+SIRPB1]; [MYADM+SLC9A1]; [MYADM+ITGA5];        [MYADM+CD70]; [MYADM+KCNN4];    -   [PIEZO1+SIRPB1]; [PIEZO1+SLC9A1]; [PIEZO1+ITGA5]; [PIEZO1+CD70];        [PIEZO1+KCNN4];    -   [SIRPB1+SLC9A1]; [SIRPB1+ITGA5]; [SIRPB1+CD70]; [SIRPB1+KCNN4];    -   [SLC9A1+ITGA5]; [SLC9A1+CD70]; [SLC9A1+KCNN4];    -   [ITGA5+CD70]; [ITGA5+KCNN4] and    -   [CD70+KCNN4].

The 32 rank selections yeilded total 496 pairs of targets, which areshow in Table 1.

A combinatorial targeting and recognition strategy was previouslydeveloped with T cells transduced with both a suboptimal CAR and achimeric co-stimulatory receptor (CCR) recognizing a second antigen.Co-transduced T cells destroy cells expressing both antigens but do notaffect cells expressing either antigen alone (Kloss et al., 2013). Thisstrategy are to be employed to assess and compare the efficacy andspecificity of the pairs of CARs in AML (FIG. 1E). Exemplary combinationcan be: LTB4R1+CD70; CD70+EMR2; and LTB4R1+EMR2.

Further validation by flow-cytometry in large panel of AML cells andadditional optional validation (in genetically-defined subsets of AMLcells) refined the choice of the top target or pair of targets and/orindicate the best CAR treatment option based on the specific mutationalprofile of AML (FIG. 1B—step#6 and #6′).

2. Antigen Expression Levels in Normal and Malignant Cells byFlow-Cytometry

The expression levels of top 11 antigens were accessed in normal CD34+,CD34+CD38− HSCs, CAR+ T cells (before and after activation) and in fourmalignant AML (THP1, Monomac, Molm13 and 09AML) cells.

FIG. 2A shows 9 candidates: LTB4R, EMR2, SLC9A1, MYADM, CD33, SLC6A6,KCNN4, PIEZO1, SIRPB1. LTB4R and EMR2 present the best expressionprofile compared to the whole panel in FIG. 2.

FIG. 2B shows 3 candidates: CD70, ENG, ITGA5. These antigens share highexpression in T cells.

FIG. 2C shows 13 candidates: CCR1, SLC22A5, TFR2, LILRB4, GYPA, FCGR1A,IL10RB, PLXNC1, CD300LF, MBOAT7, MRP1, SLC43A3, SLC44A1. These antigensshare a non-homogenous expression in all AML cells.

FIG. 2D shows 6 candidates: CPM, TTYH3, ITGA4, SLC19A1, CD38, ICAM1.These antigens share high expression in normal HSCs.

The top 11 antigens were used to screen for combanatorial CAR targets asdiscussed above, which yeilded 55 pairs of suitable targets (FIG. 3A).FIG. 3B shows the flow cytometry results verifying expression of LTB4R1and EMR2, one pair of the 55 pairs of targets, in normal and malignantcells. FIG. 4 depict the combinatorial approach. Using CD70-CCR andCD33-CAR as example, the CAR and CCR can be constructed in separatevector/cassette (the upper two schematics), or in a bicistronic cassette(the bottom schematic, with CD33 CAR and CD70 CCR linked by a 2Apeptide). The expression of the CD33 CAR and CD70 CCR in T cells and thecytotoxic T lymphocyte (CTL) assay in AML cells are also shown in FIG.4.

Information on top single targets (also included in the combinatorialstudy) is provided below. They both belong to the family of Gprotein-coupled receptors (GPCRs). GPCRs represent the largest family ofmembrane receptors with an estimated number of 800 members in human.Several GPCRs are critical for cell proliferation and survival and canbe aberrantly expressed in cancer cells (O'Hayre et al., 2014). In AML,for instance CXCR4 overexpression has been associated with poor outcome(Konoplev et al., 2007). RNA-seq analysis of GPCRs in 148 AML samples vsnormal hematopoietic cells demonstrated that the most highly expressedGPCRs in AML cells are in decreasing order: CXCR4, CD97, PTGER4, GPR183,PTGER2, S1PR4, FPR1, EMR2, C3AR1, LTB4R, TPRA1, C5AR1, LPAR2, LTB4R2 andGPR107 (Maiga et al., 2016). Chemokine receptors found to beoverexpressed in AML specimens such as CCR1 have a crucial role in thepathogenesis of myeloma-associated bone disease and CCX721, a selectiveCCR1 inhibitor, improves osteolytic bone lesions in a preclinical mousemodel of this disease (Dairaghi et al., 2012). This suggests that thesesurface receptors are potential novel therapeutic targets in AML.

Leukotriene B4 (LTB4), an eicosanoid derivative of arachidonic acidmetabolism produced by the sequential action of 5-lipoxygenase andleukotriene A4-hydrolase, is a leukocyte chemoattractant (Samuelsson etal., 1987). LTB4 signals through two G protein-coupledseven-transmembrane domain receptors, LTB4 receptor (BLT1) and BLT2, thehigh- and low-affinity receptors, respectively (Yokomizo et al., 1997;Yokomizo et al., 2000). BLT1 is a mediator of inflammation (Kim et al.,2006). In sharp contrast to BLT2, which is expressed ubiquitously, BLT1is predominantly expressed in granulocytes. Yokota et al. used aGM-CSF-based tumor vaccine setting in BALB/c leukemia model and showedbetter primary and recall immune responses in the BLT1−/−mice (Yokota etal., 2012). In human promyelocytic leukemia cell lines (HL60), theexpression of BLT1 during neutrophilic differentiation is markedlyincreased by stimulation with retinoic acid (RA)(Obinata et al., 2003).An enhancer element, termed AE-Blex, is involved in the facilitation ofBLT1 expression in leukocytes. AE-BLex contains 2 recognition sites forAML1 (aka Runxl), both of which are required for the enhancer function.The histone acetylation and chromatin remodeling of this region arefacilitated during the neutrophilic differentiation of leukemia cells,providing access for AML1 and other transcription factors (Hashidate etal., 2010). Sp1 site is the essential element of the BLT1 promoter (Katoet al., 2000). Ohler et al identified LTB4R as a top gene (out of 2612differentially expressed genes) able to discriminate blastic crisis fromchronic phase CML by applying a probabilistic method called Bayesianmodel averaging (BMA) to a large CML patient microarray dataset (Oehleret al., 2009).

In addition to mediating the chemotactic responses to LTB4 inleukocytes, the LTB4 receptor has been shown to act as a co-receptormediating entry of HIV-1 into CD4-positive cells (Martin et al., 1999;Owman et al., 1998). Thus, a CAR targeting this receptor may be utilizedalso for this purpose. LTB4R was detected on peripheral blood monocytes,lymphocytes and granulocytes by flow-cytometry (Dasari et al., 2000).Human BLT1 protein expression has been confirmed by flow cytometry usinganti-BLT1 monoclonal antibodies on CD15+ peripheral blood granulocytes,and on HL-60 cells when differentiated into neutrophil-like cells bytreatment with DMSO (Pettersson et al., 2000). The human high-affinityLTB4 receptor was cloned by Yokomizo et al in 1997 from retinoicacid-differentiated HL-60 cells using a subtraction strategy (Yokomizoet al., 1997). BLT1 binds to LTB4 with significantly greater specificitythan BLT2. Multiple LTB4 receptor anatgonists have been developed,including CP-105,696 (Showell et al., 1995), CP-195,543 (Showell et al.,1998), U-75302 (Lawson et al., 1989), ZK158252 and ONO-4057 (Kishikawaet al., 1992). Some of these agents selectively antagonize BLT1, whereasothers antagonize both receptors. Leukocyte BLT1 expression isupregulated in inflammation. Specific inflammatory stimuli responsiblefor the induction of BLT1 expression have not yet been fullycharacterized, but to date both IFNgamma and glucocorticoids have beenshown to induce BLT1 expression.

EMR2 is a member of the epidermal growth factor (EGF)-TM7 family ofproteins. EMR2, EMR1 (EGF-like molecule containing mucin-like hormonereceptor 1)(Baud et al., 1995), F4/80 (the probable mouse homologue ofhuman EMR1)(Lin et al., 1997) and CD97 (Hamann et al., 1995) constitutethe class B GPCR subfamily and are predominantly expressed onleukocytes, suggesting a role in the immune system by interacting witheither cell surface proteins or extracellular matrix proteins, possiblyleading to signal transduction via the 7TM domain. These moleculespossess N-terminal EGF-like domains coupled to a seven-spantransmembrane (7TM) moiety via a mucin-like spacer domain. EMR2 sharesstrikingly similar molecular characteristics with CD97. It maps closelyto CD97 on human chromosome 19p13.1 region (contains 20 exons), andcontains a total of five tandem highly homologous EGF-like domains,indicating that both genes are the products of a gene duplication event.The EGF domains of EMR2 are almost identical to those of CD97 with thesequence identity ranging from 95 to 100% in corresponding EGF domains.Of 236 amino acid residues in the five EGF domains of EMR2 and CD97,only 2 residues in domain 1, 1 residue in domain 2, and 3 residues indomain 3 are different. The high degree of identity in the EGF domainsconserves the consensus amino acid sequences for the EGF domaincalcium-binding site and for posttranslational beta-hydroxylation ofaspartate/asparagine, which were identified in EGF domains 2-5 (Gray etal., 1996; Hamann et al., 1995). Such calcium-binding EGF domains, foundin a broad spectrum of extracellular proteins have been shown to play animportant role in protein-protein interactions involving cell adhesion,blood coagulation and receptor-ligand binding (Downing et al., 1996),including the interaction of CD97 and its cellular ligand CD55 (Hamannet al., 1998). Significant amino acid sequence homology between EMR2 andCD97 also extends to the spacer region (46% identity) and the 7TM region(45% identity). Multiple potential N- and O-glycosylation sites withinthe extracellular domain are found conserved as well. Monoclonalantibodies (mAbs) raised against the extracellular spacer domain of CD97are able to differentiate these two proteins. Within the spacer region,a cysteine-rich motif of approximately 55 amino acids locatedimmediately before the first TM segment was also recognized; this motifis characterized by four invariant cysteine residues and two conservedtryptophan residues and is found in other members of the EGF-TM7 familyand family B GPCR-related proteins. The cysteine-rich motif, named GPSfor GPCR proteolytic site, is believed to be involved in the proteolyticcleavage. In addition four consensus sequences for protein kinaseC-mediated phosphorylation in intracellular loops 2 and 3 and thecytoplasmic tail were identified. Unlike CD97, which is ubiquitouslyexpressed in most cell types, EMR2 expression is restricted tomonocytes/macrophages and granulocytes. In addition, CD97 is rapidlyup-regulated in activated T and B cells but similar up-regulation is notobserved for EMR2 (Lin et al., 2000). EMR2 fails to interact with CD55,the cellular ligand for CD97 (Hamann et al., 1998) and may thereforehave a unique function in the myeloid lineage (Lin et al., 2000). EMR2protein is a cell surface molecule containing a long N-terminalextracellular region of 511 amino acids, a 7TM region of 248 amino acidsand a cytoplasmic tail of 41 amino acids.

Alternative splicing has been found to occur predominantly at the 5′-endof the transcripts, potentially resulting in multiple protein isoformsthat contain different numbers and/or combinations of EGF-like domains(Gray et al., 1996; Lin et al., 1997; McKnight and Gordon, 1996).Putative EMR2 protein isoforms containing five EGF domains (EGF 1-5),four EGF domains (EGF 1,2,3,5), three EGF domains (EGF 1,2,5), and twoEGF domains (EGF 1,2) are predicted. A splice variant resulting from theby-pass of exon 12 predicted to encode a soluble EMR2 molecule. Possibleligand candidates include the regulators of complement activation, suchas CD46, CD35, CD21 and C4-binding protein, all of which contain shortconsensus repeats similar to those found in CD55 (Liszewski et al.,1996).

CD70 is the membrane-bound ligand of the CD27 receptor, which belongs tothe tumor necrosis factor receptor superfamily (Bowman et al., 1994;Hintzen et al., 1994). CD70 is expressed by DLBCL, follicular lymphoma,Hodgkin lymphoma (Lens et al., 1999), Waldenstrom macroglobulinemia,multiple myeloma, human T-lymphotropic virus type 1-(Baba et al., 2008),EBV-associated malignancies (Agathanggelou et al., 1995), renal cellcarcinoma (Junker et al., 2005) and glioblastoma (Chahlavi et al.,2005). Physiologically, CD70 expression is transient and restricted to asubset of highly activated T, B, and dendritic cells. TargetingCD70-positive malignancies with CD70-specific monoclonal antibodies hasshown promise in preclinical animal models (McEarchern et al., 2007;McEarchern et al., 2008) and Shaffer et al have targeted CD70 generatingCD70-specific CAR, consisting of full-length CD27 as the antigenrecognition domain fused to the intracellular domain of the CD3-zetachain (Shaffer et al., 2011).

Generation of Human Genetic Models of Pre-Leukemic Stem Cells

In order to design a curative therapeutic strategy with CAR T cells, thetop candidates in leukemic initiating cells are to be further validated.To this purpose, key epigenetic mutations in CD34⁺ HSPCs, that werepurified from cord blood, were retrovirally expressed (Perna et al.,2010). Pre-leukemic stem cells are genetically defined by the expressionof initiating mutations such as DNMT3a (Shlush et al., 2014b). Severalmutant oncogenes including DNMT3aR882H were cloned into MSCV retroviralvectors carrying GFP that served as selection marker of the transducedcells. Most of the oncogenic mutations used herein also characterizespecific phenotypic subsets of AML, behave as dominant-negative on thewild type allele and associate with poor prognostic outcome, thusrepresenting the types of patients that will most likely benefit fromimmune mediated therapies.

The DNA (cytosine-5)-methyltransferase 3A (DNMT3A) is a member of theDNA methyltransferase family and one of the most frequently mutatedgenes in AML, occurring in up to 36% of cytogenetically normal AML(CN-AML) patients (Marcucci et al., 2012). Despite the high frequency ofmutations in DNMT3A in AML and their consistent association with adverseprognosis, the targets of DNMT3A mutations, which might contribute toleukemogenesis have not been definitively delineated. A recurrentheterozygous mutation at residue Arginine 882 accounts for 40% to 60% ofDNMT3A mutations (Ley et al., 2010; Yan et al., 2011). In AML cells,R882 mutations always occur with retention of the wild-type allele andit was showed that the R882 mutant serves as a dominant-negativeregulator of wild-type DNMT3A (Russler-Germain et al., 2014).Methylation studies using the HELP (HpaII tiny fragment enrichment byligation-mediated polymerase chain reaction) assay have not thus farresolved a methylation-specific signature characteristic of DNMT3Amutant AML samples compared with DNMT3A wild-type samples. DNMT3amutation is an early event in leukemogenesis. In fact, purified HSCs,progenitor and mature cell fractions from the blood of AML patientscontain recurrent DNMT3A mutations at high allele frequency andDNMT3Amut-bearing HSCs show a multilineage repopulation advantage overnon-mutated HSCs in xenografts. These finding established theDNMT3amut-expressing HSCs are pre-leukemic HSCs. Pre-leukemic HSCs werefound in remission samples, indicating that they survive chemotherapy,leading to a clonally expanded pool of pre-leukemic HSCs from which AMLevolves (Shlush et al., 2014a). Currently, no specific therapiestargeted toward DNMT3A have been developed to date.

Rearrangements of the Mixed-Lineage Leukemia (MLL) gene are foundin >70% of infant leukemia, ˜10% of adult AML, and many cases ofsecondary acute leukemias. The presence of an MLL rearrangementgenerally confers a poor prognosis. The most common fusion proteinMLLAF9 induces the inappropriate expression of homeotic (Hox) genes,which, during normal hematopoiesis, are maintained by wild-type MLL.Studies in mice have demonstrated that MLL-fusions can conferself-renewal activity to committed myeloid progenitors (Cozzio et al.,2003; So et al., 2003).

The AML1-ETO fusion transcription factor is generated by the t(8; 21)translocation, which is present in approximately 4%-12% of adult and12%-30% of pediatric AML patients. Both human and mouse models of AMLhave demonstrated that AML1-ETO is insufficient for leukemogenesis inthe absence of secondary events. Although AML patients harboring thet(8; 21) translocation are generally given a good prognosis and themajority achieve complete remission, the 5-year survival is only ˜50%,and the presence of a c-kit mutation decreases the prognosissignificantly. The identification of novel therapeutic targets in t(8;21) positive AML may lead to treatment options that improve patientsurvival.

The t(15; 17)(q24; q21), generating a PML-RARA fusion gene, is thehallmark of acute promyelocytic leukemia (APL). The resulting fusionprotein retains domains of the RARA protein allowing binding to retinoicacid response elements (RARE) and dimerization with the retinoid Xreceptor protein (RXRA). They participate in protein-proteininteractions, associating with RXRA to form hetero-oligomeric complexesthat can bind to RARE. They have a dominant-negative effect on wild-typeRARA/RXRA transcriptional activity. Moreover, RARA fusion proteins canhomodimerize, conferring the ability to regulate an expanded repertoireof genes normally not affected by RARA. RARA fusion proteins behave aspotent transcriptional repressors of retinoic acid signaling, inducing adifferentiation blockage at the promyelocyte stage, which can beovercome with therapeutic doses of ATRA or arsenic trioxide. However,resistance to these two drugs is a major problem, which necessitatesdevelopment of new therapies.

Primary CD34+ HSCs isolated from cord blood were retrovirally infected,and a “myeloid priming” was provided in liquid cultures supporting themyeloid differentiation. The GFP+ cells were sorted by FACS and used forflow-cytometry, Mass-Spect and RNA-seq analyses. Cell surface geneslisted in FIG. 5A have at least 2-fold increase in the DNMT3a R882Hmutant cells compared to control cells (MIGR1). Cell surface geneslisted in FIG. 5B have at least 2-fold increase in the MLLAF9 mutantcells compared to control cells (MIGR1). Those genes (also listed inTable 2) can be considered for CAR target in AML therapy.

Conclusions

CAR therapy is a novel approach to cancer immunotherapy that hasdemonstrated great potential against B-cell malignancies and may soon beapproved for relapsed, chemo-refractory ALL. One may anticipate asimilar outcome for AML if suitable targets are identified.

Through an innovative multi-tiered platform integrating surface-specificproteomics and transcriptomics in several malignant and normal cellsubsets, 32 candidates, 11 top candidates, 4 candidates for single CARstrategies and 55 pairs, 3 top pairs of targets for combinatorialstrategies were identified. Furhtermore, pre-leukemic stem cells modelindicated an additional set of surface genes suitable for CAR therapy

Materials and Methods

Flow-Cytometry

We used the following antibodies to define antigen expression byflow-cytometry:

-   -   CD70-PE cat.355104 (Biolegend);    -   EMR2-FITC cat.130-104-654; EMR2-APC cat. 130-104-656 (Milteny);    -   LTB4R1-AF700 cat.FAB099N; LTB4R1-AF405 cat.FAB099V; LTB4R1-FITC        cat.NB100-64832 (Novus Biologicals); LTB4R1-PE cat. FAB099P        (R&D)    -   PIEZO1-AF488 cat.NBP11-78537;    -   CD33-APC cat.551378 (BD Pharmingen);    -   ENG-APC cat. MHCD10505 (Invitrogen);    -   MYADM cat.NBP2-24494SS (Novus)    -   ITGA5 (CD49e)-APC cat. 328011 (Biolegend)    -   SLC19A1-APC cat. FAB8450A (R&D)    -   ILT3-APC (LILRB4) cat. FAB24251A (R&D)    -   CCR1-PE cat. 130-100-368 (Milteniy)    -   ITGA4-APC cat. FAB2450A (R&D); CD49d-PE cat. 130-099-691        (Miltenyi)    -   ICAM1-PE cat. 130-103-909 (Miltenyi)    -   SIRPB1-PE cat. 130-105-310 (Miltenyi)    -   CD64-APC (FCGR1A) cat. 561189 (BD)    -   CD300f (IREM-1)-PE cat.130-098-472; CD300f (IREM-1)-FITC        cat.130-098-443 (Miltenyi);    -   IL10RB-APC cat. FAB874A (R&D)    -   MRP1-PE cat. IC19291P (R&D)    -   CD38− APC cat. MHCD3805; CD38− PE cat. MHCD3804 (Invitrogen);    -   CD34-APC cat. 340667 (BD)    -   CPM cat.DDX0520P (Dendritics)    -   TTYH3 cat. NBP1-91350 (Novus)    -   SLC NHE1 (SLC9A1) ab58304 (abcam)    -   SLC22A5 bs-8149R (Bioss)    -   KCNN4 PA5-33875 (Thermo Scientific)    -   ITFG3 PA5-31403 (Thermo Scientific)    -   SLC6A6 LS-C179237 (LSBio)    -   SLC43A3 NBP1-85026 (Novus)    -   TFR2 TA504592 (Origene)    -   MBOAT7 NBP1-69610 (Novus)    -   CD235a-APC (GYPA) cat. 551336 (BD Pharmigen)    -   PLXNC1 cat. AF3887-SP (R&D Systems)        Vectors Cloning

DNMT3a WT, DNMT3a R882H, IDH2 WT, IDH2 R172K, IDH1 WT, IDH1 R132C, IDH1R132H, IDH2 R140Q, IDH1 R140H were cloned into MIGR1 MSCV GFP vectors.Design in CD70 CAR and CD33 CAR are shown in FIG. 5.

CD34+ Cells Purification and Culture Conditions

Mononuclear cells are isolated by centrifugation on a gradient ofFicoll-Hypaque Plus density. CD34+ HPCs are purified by positiveselection using Midi MACS (magnetic-activated cell sorting) LS+separation columns and isolation Kit according to the manufacturer'sprotocol (Miltenyi). Cells may be frozen in DMEM supplemented with 10%DMSO and stored in liquid nitrogen.

One day before the transduction, CD34+ cells are thawed and cultured inIscove's modified Dulbecco's medium (IMDM) containing 20% BIT mediumsupplemented with SCF (100 ng/ml), FLT-3 (10 ng/ml), IL-6 (20 ng/ml) andTPO (100 ng/ml). Cytokines may be purchased purchased from Peprotech andR&D. After a 24 hours-recover CD34+ cells are infected with high-titerretroviral suspensions in presence of polybrene (8 μg/ml).

Generation and Concentration of Retroviruses

Retroviral vectors are produced by transfection of H29 packaging cellsaccording to standard protocols. Briefly H29 cells may be cultured inDulbecco modified Eagle medium (DMEM), supplemented with 10% fetalbovine serum (FBS) and when subconfluent, transfected with plasmids. Thetransfection is performed by calcium-phosphate precipitation. Vectorssupernatants are harvested 6 and 7 days later and used to infect RD114cells.

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Example 2

To rapidly obtain two fully human high-quality single-chain fragments(scFvs) targeting LTB4R1 and EMR2, in-house next-generation, premade,fully human, phage display scFv libraries is first utilized. Theadvantages include phage stability, rapid production, the fact thatintrinsic properties such as immunogenicity, affinity, specificity andstability of antibodies can be improved by various mutagenesistechnologies. The antibody genes are expressed and the gene productsdisplayed on the surface of filamentous bacteriophage as fusionproteins, thus creating a link between antibody phenotype and itsencoded genotype (Pansri, BMC Biotechnol, 9:6, 2009; Farajnia,Immunopharmacol Immunotoxicol, 36:297-308, 2014).

Example 3

To expand the validation cohort of the targets within primary AMLpatient samples, at least 50 samples, bearing frequently recurringgenetic abnormalities, including DNMT3a mutation, are analyzed by flowcytometry.

Leukotriene B4 (LTB4), an eicosanoid derivative of arachidonic acidmetabolism produced by the sequential action of 5-lipoxygenase andleukotriene A4-hydrolase, is a leukocyte chemoattractant30. LTB4 signalsthrough two G protein-coupled seven-transmembrane domain receptors, LTB4receptor (BLT1) and BLT2, the high- and low-affinity receptors,respectively (Yokomizo, Nature, 387:620-624, 1997; Yokomizo, J Exp Med,192:421-432, 2000). BLT1 is a mediator of inflammation (Kim, J Exp Med,203:829-835, 2006). In sharp contrast to BLT2, which is expressedubiquitously, BLT1 is predominantly expressed in granulocytes. Yokota etal. used a GM-CSFbased tumor vaccine setting in BALB/c leukemia modeland showed better primary and recall immune responses in the BLT1−/−mice(Yokota, Blood, 210:3444-3453, 2012).

EMR2 is a new member of the EGF-TM7 family of proteins, containing atotal of five tandem EGF-like domains and shares strikingly similarmolecular characteristics with CD97. Unlike CD97, which is ubiquitouslyexpressed in most cell types, EMR2 expression is restricted tomonocytes/macrophages and granulocytes. EMR2 fails to interact withCD55, the cellular ligand for CD97 and may therefore have a uniquefunction in the myeloid lineage (Lin, Genomics, 67:188-200, 2000).

Example 4

To assemble the antibody-targeting domains into second-generation CARs(bearing a single costimulatory element within the endoplasmic domainwith the activating CD3ζ domain) or CCR (fusion molecule couplingantigen specificity to T cell co-stimulatory signaling withoutactivating domains). The combinations of CAR/CCR are retrovirallyexpressed in T cells and the efficacy in damaging AML cells is evaluatedcompared to normal cells in a panel of representative cell lines bycytotoxic lymphocyte assays.

Example 5—Probing the Acute Myeloid Leukemia Surfaceome for ChimericAntigen Receptor Targets

Summary

Chimeric antigen receptor (CAR) therapy targeting CD19 has yieldedremarkable outcomes in patients with acute lymphoblastic leukemia. Toidentify potential CAR targets in acute myeloid leukemia (AML), the AMLsurfaceome was probed for over-expressed molecules absent from vitaltissues. Large transcriptomics and proteomics data sets were integratedfrom malignant and normal tissues, and developed an algorithm toidentify potential targets expressed in leukemia stem cells, but not innormal CD34+CD38− hematopoietic cells, T cells or vital tissues. Asthese investigations did not uncover candidate targets with a profile asfavorable as CD19, a generalizable combinatorial targeting strategyfulfilling stringent efficacy and safety criteria were developed. Thefindings indicate that several target pairings hold great promise forCAR therapy of AML.

Introduction

An ideal CAR target should be expressed at high density, in most if notall tumor cells including cancer stem cells, and in a large fraction ofpatients (Table 3). Unlike native T cells, which are known to signalthrough the TCR in response to very low antigen density, CAR T cellsrequire higher antigen densities to fully activate effector functions(Turatti et al., 2007; Walker et al., 2017; Weijtens et al., 2000). Highabsolute antigen expression that is easily detected by FACS analysis isthus much preferred for CAR target selection. Clonal heterogeneitycreates complex tumors that are prone to escape targeted therapies.Expression of the target in normal tissues may be tolerable (transientor partial elimination of non-vital cell types) or unacceptable(destruction of vital tissues, hematopoietic stem cell depletion). Toprevent undue toxicity, the ideal tumor target should not be expressedon any normal tissue/organ of, or at least not in vital tissues (heart,liver, CNS, lung and other tissues that cannot withstand transientdamage) nor in closely related normal cellular counterparts, i.e., CD34+hematopoietic stem/progenitor cells (HSPCs) in the case of AML. Thetarget antigen should also not be expressed in CAR T cells to obviatefratricide elimination (Table 3). It is therefore imperative tocarefully evaluate candidate targets not just in tumor cells, but acrossall normal tissues. Consequently, this task requires comprehensivesources of antigen annotation, as well as analytical tools specificallydesigned to identify potential CAR target antigens.

TABLE 3 Features of an ideal CAR target Goal Activity ExpressionEfficient High in all tumor cells recognition and on-tumor at high leveltargeting by CAR in many patients T cells Safe Low NOT in:discrimination of off-tumor any normal tissue, especially vital tissuestarget cells by normal counterparts (eg HSPCs for AML) CAR T cellsresting/activated T cells

To date, searches for CAR targets have largely relied on transcriptomeanalyses, under the assumption that there exists a direct correspondencebetween mRNA transcripts and protein expression. The correlation betweenmRNA and protein expression is complex and potentially unrepresentativeof the cell proteome, due to multiple factors including variablehalf-lives and post transcriptional regulatory mechanisms (Hack, 2004).An integrated and comprehensive analysis of transcriptomic and proteomicdata in both malignant and normal cells is therefore needed to captureinformation lacking from indirect analyses of mRNA or limited proteinexpression assays (Haider and Pal, 2013). Moreover, advanced proteomictechnologies combined with enrichment strategies for identifying plasmacell membrane proteins can provide direct measurements of the surfaceproteome. Both proteomic and genomic datasets were therefore integratedfrom AML and normal cell populations, including the surface-specificmass-spectrometry analyses of AML cell lines. After compiling acomprehensive dataset of antigen annotations, in both normal andmalignant cells, a multi-step ranking algorithm were developed for theidentification of potential CAR targets. These were then validated byflow cytometry in primary AML patient samples, normal bone marrow (BM)HSPCs and peripheral blood (PB) T cells. Here the most promisingcandidates and candidate pairs that meet stringent criteria for servingas prospective targets in CAR therapies targeting AML were report.

Materials and Methods

Experimental Model and Subject Details

Primary AML specimens were obtained from the Hematology Oncology TissueBank (HOTB) of MSKCC (IRB protocol Y2017P026). Patient characteristicsare illustrated in Supplemental Table 2.

Primary human bone marrow CD34+ cells were purchased from Stem CellTechnologies (70002.2, 70002.3).

Human T Cells Isolation and Activation

Buffy coats from healthy volunteer donors were obtained from the NewYork Blood Center. Peripheral blood mononuclear cells were isolated bydensity gradient centrifugation, and T lymphocytes were then purifiedusing the Pan T cell isolation kit (Miltenyi Biotech). Cells wereactivated with Dynabeads (1:1 beads:cell) Human T-Activator CD3/CD28(ThermoFisher) in X-vivo 15 medium (Lonza) supplemented with 5% humanserum (Gemini Bioproducts) with 100 U/ml IL-2 (Miltenyi Biotech) at adensity of 106 cells/ml. The beads were removed by magnetic separation48 h after activation. The medium was changed every 2 days, and cellswere replated at 106 cells/ml.

AML Cell Lines

THP1, Mono-mac, Kasumi, Molm13, OCI/AML3 and TF-1 cells were maintainedin RPMI1640/1-Glutamine (Life Technologies, Inc., Carlsbad, Calif.),supplemented with 10% FBS (20% for HL60) (Life Technologies) at 37° C.THP1 and Mono-mac lines bear MLL-AF9 translocation, the THP1 line alsobears deletion of p16, p53, UTX and rearrangement of RB1; Kasumi bearsan AML1-ETO translocation; Molm13 FLT3-ITD, OCI/AML3 a NPM mutation andTF-1 a highly rearranged hyperdiploid karyotype with p53 mutation.

Normal Tissue Proteomics Compilation and Data Retrieval

Expression data for normal tissues was retrieved from three datarepositories, the Human Protein Atlas (HPA) (www.proteinatlas.org,normal_tissue.csv.zip, accessed Oct. 15, 2016), the Human Proteome Map(HPM) (RRID:SCR_015560, www.humanproteomemap.org,HPM_gene_level_epxression_matrix_Kim_et_al_052914.csv accessed Oct. 13,2016), and the Proteomics Database (PDB) (RRID:SCR_015562, Accessed viathe PDB API, available at www.proteomicsdb.org, Oct. 13, 2016).Additionally, subcellular localization data was obtained from the HPA(RRID:SCR_006710, www.proteinatlas.org,subcellular_localization.csv.zip, accessed Oct. 15, 2016) andCOMPARTMENTS (RRID:SCR_015561, compartments.jensenlab.org,LOCATE_human_v6_20081121.xml, accessed Oct. 15, 2015) repositories, andtranscriptomic data retrieved from the HPA (www.proteinatlas.org,rna_tissue.csv.zip, accessed Oct. 15, 2015) and Bloodspot.eu dataarchives (RRID:SCR_015563).

Correcting Tissue and Organelle Nomenclature

Due to differing tissue nomenclature among source repositories, eachdata set was mapped to a set of consensus tissue labels. In cases wheremultiple tissues from one repository mapped to a single label fromanother source, the maximum expression value was taken, for instance thePDB's “retina” and “vitreous humor” tissues were collapsed into a singletissue category, “eye.” For consistency, fetal and placental tissueswere also discarded, resulting in 43 distinct tissue categories (Table4) shown below: adipose tissue, adrenal, appendix, bladder, blood, bone,brain, breast, bronchus, cerumen, cervix, epididymis, eye, fallopiantube, gallbladder, gut, heart, kidney, esophagus, liver, lung, lymphnode, nasopharynx, oropharynx, ovary, pancreas, parathyroid, prostate,rectum, seminal, skeletal muscle, skin, smooth muscle, soft tissue,spinal cord, spleen, stomach, synovial fluid, testis, thyroid, tonsil,uterus, vagina

Similarly, maps of subcellular localization labels from the HPA andCOMPARTMENTS databases were generated by manually classifying organellelabels as either cell membrane-associated or otherwise unaffiliated, andthen applying the resulting dictionary to proteins in both repositories.

TABLE 4 Consensus Tissue Name HPA HPM PDB 1 adipose tissue ‘adiposetissue’ ‘adipocyte’ 2 adrenal ‘adrenal gland’ ‘Adult.Adrenal’ ‘adrenalgland’ 3 appendix ‘appendix’ 4 bladder ‘urinary bladder’‘Adult.Urinary.Bladder’ ‘urinary bladder’, ‘urine’ 5 blood ‘B.Cells’,‘CD4.Cells’, ‘B-lymphocyte’, ‘blood’, ‘blood ‘CD8.Cells’, platelet’,‘cytotoxic T- ‘Monocytes’, lymphocyte’, ‘helper T- ‘NK.Cells’,‘Platelets’ lymphocyte’, ‘monocyte’, ‘natural killer cell’, ‘serum’ 6bone ‘bone marrow’ ‘bone’, ‘bone marrow stromal cell’, ‘mesenchymal stemcell’ 7 brain cerebellum’, ‘cerebral cortex’, ‘Adult.Frontal.Cortex’‘brain’, ‘cerebral cortex’, ‘hippocampus’, ‘lateral ventricle’‘prefrontal cortex’ 8 breast ‘breast’ ‘breast’ 9 bronchus ‘bronchus’ 10cerumen ‘cerumen’ 11 cervix ‘cervix, uterine’ ‘cervical epithelium’,‘cervical mucosa’, ‘uterine cervix’, ‘uterus’ 12 epididymis ‘epididymis’13 eye ‘Adult.Retina’ ‘retina’, ‘vitreous humor’ 14 fallopian tube‘fallopian tube’ 15 gallbladder ‘gallbladder’ ‘Adult.Gallbladder’ ‘gallbladder’ 16 gut ‘colon’, ‘duodenum’, ‘small ‘Adult.Colon’ ‘colon’,‘colon muscle’, ‘colonic intestine’ epithelial cell’, ‘gut’, ‘ileumepithelial cell’ 17 heart ‘heart muscle’ ‘Adult.Heart’ ‘heart’,‘proximal fluid (coronary sinus)’ 18 kidney ‘kidney’ ‘Adult.Kidney’‘kidney’ 19 eesophagus ‘esophagus’ ‘Adult.Esophagus’ ‘esophagus’ 20liver ‘liver’ ‘Adult.Liver’ ‘bile’, ‘liver’ 21 lung ‘lung’ ‘Adult.Lung’‘lung’ 22 lymph node ‘lymph node’ ‘lymph node’ 23 nasopharynx‘nasopharynx’ ‘nasopharynx’ 24 oropharynx ‘oral mucosa’, ‘salivarygland’ ‘oral epithelium’, ‘saliva’, ‘salivary gland’ 25 ovary ‘ovary’‘Adult.Ovary’ ‘ovary’ 26 pancreas ‘pancreas’ ‘Adult.Pancreas’‘pancreas’, ‘pancreatic islet’, ‘pancreatic juice’ 27 parathyroid‘parathyroid gland’ 28 prostate ‘prostate’ ‘Adult.Prostate’ ‘prostategland’ 29 rectum ‘rectum’ ‘Adult.Rectum’ ‘rectum’ 30 seminal ‘seminalvesicle’ ‘seminal plasma’, ‘seminal vesicle’, ‘spermatozoon’ 31 skeletalmuscle ‘skeletal muscle’ 32 skin ‘skin’, ‘skin 1’, ‘skin 2’ ‘hairfollicle’, ‘skin’ 33 smooth muscle ‘smooth muscle’ 34 soft tissue ‘softtissue 1’, ‘soft tissue 2’ 35 spinal cord ‘Adult.Spinal.Cord’‘cerebrospinal fluid’, ‘spinal cord’ 36 spleen ‘spleen’ ‘spleen’ 37stomach ‘stomach’, ‘stomach 1’, ‘stomach 2’ ‘cardia’, ‘stomach’ 38synovial fluid ‘synovial fluid’ 39 testis ‘testis’ ‘Adult.Testis’‘testis’ 40 thyroid ‘thyroid gland’ ‘thyroid gland’ 41 tonsil ‘tonsil’‘tonsil’ 42 uterus ‘endometrium’, ‘endometrium 1’, ‘myometrium’‘endometrium 2’ 43 vagina ‘vagina’Calculation of Distribution Metrics

In the interest of facilitating subsequent selection steps, expressionentries were classified into three categories: “not detected”, “low”,“medium”, and “high”, the native format in which HPA protein and RNAdata was made available. To accomplish the binning, both the HPM and PDBdatasets were first log 10 transformed, after HPM data was thentemporarily corrected for the purpose of abundance distributionestimation so as to minimize artifactual cases in which LC-MS/MS peptidefragment masses were underdetermined, resulting in multiple geneassignments. This was accomplished by collapsing protein entriesoriginating from the same experiment and occurring with precisely thesame spectral abundance measure, into a single entry during the curvefitting process (after which all entries were restored). To fit normalcurves of best fit to the observed distributions, theBroyden-Fletcher-Goldfarb-Shanno algorithm was applied (Team, 2016),after which the peak maximum and standard deviation measure was recordedfor each curve.

Gene Nomenclature Conversion

Gene identification labels for all data sources not natively provided inEnsembl gene format were then converted using the biomaRt and mygene Rpackages, as well as the Proteomics Database API to ensure comprehensivemapping between differing nomenclatures (Adam Mark, 2014; Durinck etal., 2009). In cases where multiple entries from a given data sourcemapped to the same gene ID only the highest expression value for eachtissue was retained. And in cases where entries mapped to more than oneEnsembl Gene ID, duplicate entries for each ID were made.

For each unique (protein, tissue, repository) entry, the maximumexpression value was retained and the remaining expression values, whicharose from native IDs mapped to two Ensembl Gene IDs, were discarded.

Generation of Repository Metrics

The number of available data sources for every unique (protein, tissue)entry was then recorded and the maximum binned expression abundance foreach unique (protein, tissue) entry was then computed.

Expression and Binning

Expression values within one standard deviation and above normal peakswere considered to be of “medium” (2) abundance, expression values abovethis threshold were considered of “high” (3) abundance. Similarly,expression values within one standard deviation and below the normalpeak were considered of “low” (1) abundance, and abundance valuesfalling below one standard deviation considered to have an expressionlevel low enough to consider “not detected” (0), for the purpose ofepitope selection.

AML/Normal HSPCs Ratio Analysis

The experiment-normalized RNA data obtained from Bloodspot.eu wasexponentiated using a base of two as the data was natively provided inlog 2 format, after which the maximum values taken for each unique(gene-cell type) entry. Data was then divided into two groups, AML andnormal HSPCs, with the AML group consisting of the following subtypes:−5/7(Q), −9Q, 7, 8, ABN(3Q), COMPLEX, COMPLEX_ DEL(5Q), COMPLEX_UNTYPICAL, DEL(5Q), DEL(7Q)/7Q-, DEL(9Q), INV(16), INV(3), nan, NORMAL,OTHER, T(1; 3), T(11Q23)/MLL, T(6; 9), T(8; 16), T(8; 21), T(9; 11),T(9; 22), TRISOMY 11, TRISOMY 13, and TRISOMY 8. The normal HSPCs groupconsisting of the following cell types: HSC, MPP, CMP, GMP, and MEP. Amean expression value for each gene in the AML group was then calculatedby averaging across the five normal cell types. The resulting meanvalues were then taken as devisors for expression ratios in which thedividends were each cell type's RNA abundance. The base ten logarithmwas then taken for each expression ratio and normal curves were fit tothe observed distribution using the Broyden-Fletcher-Goldfarb-Shannoalgorithm, after which the peak maximum and standard deviation measurewas recorded for each curve. A threshold of two standard deviationsabove the distribution maximum was then applied, and any proteincandidate with a ratio above this threshold recorded for later use.

Target Selection

Step 0—Surface Proteomics

Only proteins found during surface-biotinylation proteomics assaysperformed on six AML cell lines, THP1, Mono-mac, Kasumi, Molm13,OCI/AML3 and TF-1, those reported by Strassberger et al., or a selectlist of 359 previously reported molecules, including CLEC12A, IL3RA,FOLR2, FUT3, CD33, and CD38 were kept for further study.

Step 1—RNA

Further, exclusively molecules whose ratio of RNA expression in AMLsamples versus normal HSPCs cells was greater than or equal to 2 SDabove the mean were retained.

Step 2—QC

Only protein entries reported only in two or more normal tissueproteomics databases were retained for further study. Additionally,proteins were discarded from further consideration if the locationsreported in either the HPA or COMPARTMENTS databases were not on thecell membrane.

Step 3—Exclude High Expressors

Any protein entry whose mean expression across all normal tissuesexceeded the classification threshold as a “medium” (2) expressor wasexcluded from further study. Further, all proteins whose expression wasclassified as high (3) for any tissue type, in any dataset, apart fromthose originating from blood and bone were excluded from further study.

Combinatorial Selection

Pairwise Exclusion

9 candidates, in addition to CLEC12A, IL3RA and CD33, were then assessedas combinatorial pairs by evaluating their expression each tissue sitesat once. Vital and non-vital tissues were then assessed using distinctcriteria, which all tissues possessing a given protein were required topass. Due to their high concentration of hematopoietic cells, theappendix, bone, blood, and spleen tissues were not considered for thepurposes of selection. Criteria for vital tissues (adipose tissue,adrenal, bladder, brain, bronchus, eye, gut, heart, kidney, esophagus,liver, lung, nasopharynx, oropharynx, pancreas, rectum, skeletal muscle,skin, smooth muscle, soft tissue, spinal cord, and stomach) required atleast one of the tissue pairs to possess no detectable expression.Criteria for non-vital tissues (breast, cerumen, cervix, epididymis,fallopian tube, gallbladder, lymph node, ovary, parathyroid, prostate,seminal, spleen, synovial fluid, testis, thyroid, tonsil, uterus, andvagina) permit tissue expression in both antigens in a pair to exhibit“low” expression, or to possess no detectable expression, as qualifiedabove.

Surface Proteomics

Cell surface biotinylation of six (THP1, Mono-mac, Kasumi, Molm13,OCI/AML3 and TF-1) human AML cell lines were performed, which was usedfor mass spectrometric analysis.

For the isolation and collection of surface proteins, the Pierce® CellSurface Protein Isolation Kit #89881 (Thermo Scientific 89881) wereused. 6×10⁶ cells were cultured in 75 cm² flasks. Prior to surfaceprotein biotinylation, all reagents were cooled to 4° C. The cells werewashed four times with ice-cold phosphate buffered saline (PBS) followedby incubation with 0.25 mg/mL Sulfo-NHS—SS-Biotin in 10 mL ice-cold PBSper flask on a rocking platform for 30 minutes at 4° C. Thebiotinylation reaction was quenched by adding 500 μL of the providedquenching solution (Pierce). Centrifuge cells at 500×g for 5 minutes anddiscard supernatant. Cells were washed with ice-cold PBS, harvested bygentle scraping and pelleted by centrifugation. The cells were lysedusing the provided lysis buffer (Pierce) containing a protease inhibitorcocktail (Sigma) for 30 minutes on ice with intermittent vortexing.Lysates were centrifuged at 16,000×g for 2 minutes at 4° C. Theclarified supernatant was used for purification of biotinylated proteinson NeutrAvidin Agarose. Before use, 500 μL of NeutrAvidin Agarose slurrywas washed three times with Pierce wash buffer in a provided column(Pierce). The clarified supernatant was added to the slurry andincubated for 2 h at room temperature in the closed column using anend-over-end tumbler to mix vigorously and allow the biotinylatedproteins to bind to the NeutrAvidin Agarose slurry. Unbound proteinswere removed by repetitive washing; three times with 500 μL Pierce WashBuffer in a provided column (Pierce), three times with 500 μL (50 mMAmmonium bicarbonate) and eight times with 500 μL digestion buffer (50mM Tric-C1, pH 7.5, 1 mM CaCl₂). Finally bounded proteins onbiotin-NeutrAvidin Agarose were digested with 4 μg of trypsin (preparedin digestion buffer) over night at 37° C. in shaking incubator (˜750rpm). The next day, digested peptides were filtered through column andprotease reaction was stopped by of 0.5% TFA. Samples were cleared bycentrifuging 10 min at 14,000×g, 15° C. and desalted by stage tips.Desalted peptides were dry down by speed vac and re-suspended in 10 μlof 3% acetonitrile/0.1% formic acid for LC-MS/MS analysis.

LC-MS/MS Analysis

Desalted peptides were dissolved in 3% acetonitrile/0.1% formic acid andinjected onto a C18 capillary column on a nano ACQUITY UPLC system(Water) which was coupled to the Q Exactive mass spectrometer (ThermoScientific). Peptides were eluted with a non-linear 200 min gradient of2-35% buffer B (0.1% (v/v) formic acid, 100% acetonitrile) at a flowrate of 300 nl/min. After each gradient, the column was washed with 90%buffer B for 5 min and re-equilibrated with 98% buffer A (0.1% formicacid, 100% HPLC-grade water) for 4 min. MS data were acquired with anautomatic switch between a full scan and 10 scan data-dependent MS/MSscan (TopN method). Target value for the full scan MS spectra was 3×10⁶charges in the 380-1800 m/z range with a maximum injection time of 30 msand resolution of 70,000 at 200 m/z in profile mode. Isolation ofprecursors was performed with 2.0 m/z. Precursors were fragmented byhigher-energy C-trap dissociation (HCD) with a normalized collisionenergy of 27 eV. MS/MS scans were acquired at a resolution of 17,500 at200 m/z with an ion target value of 5×10⁴ maximum injection time of 60ms and dynamic exclusion for 60 s in centroid mode.

Protein Identification

MS raw files were converted into MGF by Proteome Discover (ThermoScientific) and processed using Mascot 2.4 (Matrix Science, U.K.) bysearching against the Uniport human Database (version 2014 with 20209protein entries) supplemented with common contaminant proteins. Searchcriteria included 10 ppm mass tolerance for MS spectra, 0.8 Da masstolerance for MS/MS spectra, a maximum of two allowed missed cleavages,fixed carbamidomethylation of cysteine modifications, variablemethionine oxidation and N-terminal protein acetylation, Mascotsignificance threshold of 0.05, and a false discovery rate of <0.01.Mascot data were assembled by Scaffold and X!-Tandem software and searchcriteria for identification 2 minimum peptides and 1% FDR at thepeptide, and protein level.

Flow-Cytometric Analysis

The following antibodies were used to define antigen expression byflow-cytometry:

CD82-PE (Biolegend, 342103); CD120b (TNF-RII)-PE (Miltenyi,130-107-740); EMR2-FITC, −APC (Miltenyi, 130-104-654, 130-104-656);ITGB5-PE (Biolegend, 345203); CCR1-PE, −APC (Miltenyi, 130-100-367,130-100-358); CD96-APC (Miltenyi, 130-101-031); PTPRJ (CD148)-PE (lifetechnologies, A15799); CD70-PE, −FITC (Biolegend, 355104, 355106); CD85d(ILT4)-PE −APC (Miltenyi, 130-100-567, 130-100-559); LTB4R1-AF700 (NovusBiologicals, FAB099N); CD85h (ILT1)-APC (Miltenyi, 130-100-920);TLR2-APC (Miltenyi, 130-099-020); CR1 (aka CD35)-APC (Miltenyi,130-099-923); ITGAX (CD11c)-APC (Biolegend, 301613); EMB (abcam,179801); EMC10 (abcam PA5-25112); LILRB3-PE (Miltenyi, 130-101-662);LILRB4-APC (R&D, FAB24251A); DAGLB (abcam, PA5-26331); P2RY13 (Novus,NBP2-37382); LILRA6-APC (Miltenyi, 130-101-665); SLC30A1 (Alomone labs,AZT-011); SLC6A6 (LSBio, LS-C179237); SEMA4A (R&D, FAB4694A); CD123-PE(BD Biosciences, 555644); CLEC12A-PE (Miltenyi 130-106-482); CD33-APC(Miltenyi, 130-098-864); CD38− BV421 (BD Biosciences, 562444);CD34-PE/Cy7 (Biolegend, 343515); CD45RA-BV640 (Biolegend, 304135);CD90-FITC (BD Biosciences, 555595).

Quantification and Statistical Analysis

Student's t-test was used for significance testing in the bar graphsusing a two-sample, normally distributed equal-variance model. P valuesless than 0.05 were considered to be significant. Graphs and error barsreflect means and standard deviations. All statistical analyses werecarried out using GraphPad Prism 4.0 and the R statistical environment.(*) 0.03, (**) 0.0021, (***) 0.0002, (****) 0.0001.

Allowing for a 20% margin, a sufficient single-tailed estimate ofarbitrarily large population size can be assessed at 95% confidence with23 patients. A sample size of 30 was chosen to further narrow the windowof uncertainty.

Data and Software Availability

Proteomic data were submitted (ProteomeXchange Submission) to PRIDEdatabase on Jul. 31, 2017. A temporary ticket has been assigned[px-submission #204296].

Results

Assembling a Comprehensive Dataset ofAML Surface Molecule Annotations

To search for potential CAR targets, surface-specific proteomic studieswere first performed in a diverse panel of AML (THP1, Mono-mac, Kasumi,Molm13, OCI/AML3 and TF-1) cell lines. After biotinylating the cellsurface (FIG. 11), mass-spectrometric analysis were performed and 4,942proteins were identified. In order to generate the largest possibleinclusive data set, the findings of surface-specific proteomic studiesconducted in other human myeloid leukemia lines (NB4, HL60, THP1,PLB985, K562) (Strassberger et al., 2014) and all previously reportedsurface proteins, such as CD123 (Jordan et al., 2000), CLL1 (Bakker etal., 2004), CD33 (Taussig et al., 2005), CD44 (Jin et al., 2006), CD96(Hosen et al., 2007), CD47 (Majeti et al., 2009), CD32 and CD25 (Saitoet al., 2010), TIM3 (Kikushige et al., 2010), CD99 (Chung et al., 2017)were further added to this list, adding another 80 proteins (FIG.6—orange boxes).

To annotate the expression of these molecules in normal tissues, datafrom the Human Protein Atlas (HPA) (Uhlen et al., 2015), the HumanProteome Map (HPM) (Kim et al., 2014) and the Proteomics Database (PD)(Wilhelm et al., 2014) were integrated. These data sources providedprotein expression information for several normal tissues/organs,including liver, gallbladder, pancreas, stomach, gut, duodenum, colon,rectum, testis, epididymis, prostate, breast, vagina, uterus, ovary,skin, skeletal and smooth muscle, cerebral cortex, hippocampus, lateralventricle, cerebellum, thyroid, bronchus, lung, heart, retina, vitreoushumor, bone marrow, lymphocytes, lymph nodes, tonsil, synovial fluid andothers, listed in Table 4. Data in the atlases were obtained byantibody-based immunohistochemistry (HPA) or protein Mass Spectrometry(HPM and PD) (FIG. 6—green boxes). To focus the study on moleculesspecifically annotated to the membrane, two subcellular localizationdata sources, the HPA's subcellular annotation and the Jensen Lab'sCompartments repository were relied on (FIG. 6—yellow boxes).

To remove surface molecules that are not over-expressed in AML cellscompared to normal counterparts, publicly available gene expressionanalyses in normal bone marrow CD34+CD38− CD90+CD45RA-HSCs andLin-CD34+CD38− CD90-CD45RA-multipotent progenitors (MPP),Lin-CD34+CD38+CD45RA-CD123+ common myeloid progenitor cells (CMP),Lin-CD34+CD38+CD45RA+CD123+ granulocyte monocyte progenitors (GMP),Lin-CD34+CD38+CD45RA-CD123-megakaryocyte-erythroid progenitor cells(MEP) from healthy donors (GSE42519) and 3,097 primary AML patientsamples clustered in 26 distinct subtypes based on specific cytogeneticssuch as del5q, t(8; 21), t(11q23)/MLL, inv(16)/t(16; 16) etc (GSE13159,GSE15434, GSE61804, GSE14468 and the Cancer Genome Atlas) (Bagger etal., 2016) were utilized (FIG. 6—pink boxes). In the final stage of thestudy, the expression of candidate targets selected by the algorithmswere characterized and described below, in a panel of 30 primary AMLpatient samples based on flow-cytometric analyses (FIG. 6A—blue box).This compilation (FIG. 6—grey box) represents the most comprehensiverepository of AML surface protein annotation assembled to date.

Design of an Algorithm to Identify CAR Targets

Starting from an annotated dataset of 23,118 Ensembl gene entries(19,876 unique HUGO gene identities) including 4,943 surface molecules,an algorithm to select CAR targets were designed (FIG. 7A). Given thatan ideal target should be over-expressed in tumor cells compared tonormal tissue counterparts, a log 10 expression ratio were firstcomputed between AML cells and normal HSPCs per molecule by comparingRNA expression levels in 26 genetically defined subtypes of AML, tonormal BM CD34+CD38− CD90+CD45RA-HSCs, MPP, CMP, GMP, and MEP progenitorcells. A mean expression value for each molecule in both malignant andnormal groups was calculated and a normal distribution fit to theAML/normal HSPCs ratios. A threshold of two standard deviations abovethe distribution peak maximum was applied, leaving 823 Ensembl geneentries corresponding to 682 unique HUGO entries. Antigens were thenprioritized with a membrane-associated sub-localization and redundantprotein expression data in at least 2 of the 3 databases (HPA, HPM andPDB) that annotate expression levels in normal tissues. This “qualitycontrol” step further removed 321 molecules, leaving us with 361candidates.

To eliminate any molecule highly expressed in normal tissues, proteinexpression data in normal tissues from HPA, HPM and PDB were merged,ranging from 0 (below the level of detection) to 3 (high) (FIG. 12).Molecules exhibiting high average expression (>2) across all normaltissues as well as molecules exhibiting high expression (3) in anynormal tissue were excluded, except for blood and bone/bone marrow. Withthis algorithm, 24 molecules overexpressed in AML vs. their normalcounterparts were identified and with no high expression in clusters ofnormal tissues, except for blood and bone marrow (FIG. 7B). Furtherexclusion of the spleen would additionally include CD33 and CLEC12Aamongst the top 24 CAR candidate targets (FIG. 14).

Expression Analyses in Primary AML Samples and Normal HematopoieticCells

Expression levels of the 24 candidates were analyzed by flow cytometryin 30 primary specimens of relapsed AML, enriched for AML harboringgenetic abnormalities predisposing to clinical relapse (Table 4). Thesesamples bear frequently recurring genetic abnormalities, includingmutations in DNMT3A (14), CEBP α (12), IDH2 (11), FLT3-ITD (9), NPM1(7), IDH1 (7), WT1 (4), RUNX1 (4), ASXL1 (4), SUZ12 (3), KRas (2), TET2(2), p⁵³ (1) and CBL (1). Nine of the 24 candidate targets were presentin all analyzed patient specimens and detected in >75% cells: CD82,TNFRSF1B (aka CD120b), ADGRE2 (aka EMR2 or CD312), ITGB5, CCR1 (akaCD191), CD96, PTPRJ (aka CD148), CD70, and LILRB2 (aka CD85d). In theanalyses CD123 (IL3RA), CLEC12A (CLL1) and CD33 were also included, asthese molecules are targets in current AML clinical trials. Theseantigens were also found to be expressed in >75% of AML cells in allpatients (FIG. 8A).

As an ideal CAR target should be expressed in leukemic stem cells (LSCs)(Table 3), the expression of our selected markers in AML CD34+CD38−cells were further examined using flow cytometry. All 9 targets werealso highly expressed (>75%) in this essential AML cell subset (FIG.8B). Their mean expression levels (range 78-99%) were comparable to thatof CD123 (mean 82%) and slightly higher than that of CLEC12A (mean 77%)and CD33 (mean 77%) in LSCs. Flow-cytometric analyses in normal BMCD34+CD38− CD90+CD45RA-HSCs and CD34+CD38+ progenitor cells showed that6 out of 9 molecules (TNFRSF1B, ADGRE2, CCR1, CD96, CD70 and LILRB2)were expressed at low levels (<5%) in normal HSPCs (FIG. 8C). CD123,CLEC12A and CD33 were present at higher levels in these normalprogenitor cells (9%, 20% and 8%, respectively) (FIG. 8C).

As CAR therapy requires the sustained activity of functional CAR Tcells, expression of these antigens in freshly purified and activated Tcells from healthy donors were investigated. Four of the latter 6candidates (ADGRE2, CCR1, CD70 and LILRB2) showed low-level expression(<5%) in T cells (FIG. 8D). TNFRSF1B and CD96 were more abundant (up to67% and 83%, respectively), which may complicate the generation oractivity of CAR T cells and would require adapted strategies (e.g.,target gene ablation) if one were to pursue these antigens in a CARtherapy.

In summary, the selection process identified 4 potential CAR targetswith high expression in AML bulk cells and AML LSCs (FIG. 8A-B) and lowexpression in normal tissues (FIG. 7B), normal HSPCs (FIG. 8C) andresting/activated T cells (FIG. 8D), as depicted in FIG. 8E. Theseexpression profiles compare favorably with CD123, which is highlyexpressed in AML, especially LSCs (FIG. 8A-B), but is also abundant inmultiple normal tissues (FIG. 7B). CD33 and CLEC12A are also highlyexpressed in AML (FIG. 8A), although they exhibit a high degree ofexpression in normal hematopoietic progenitor cells (FIG. 8C),consistent with their RNA expression levels (Bagger et al., 2016).Integrated systemic proteomics data indicate that CD33 is more abundantin the lung, prostate and skin (FIG. 7B).

It is noteworthy that none of these molecules showed a profilecomparable to that of CD19, which is expressed at high levels invirtually all B cell leukemia cells, remains completely absent fromHSPCs and T cells, and undetectable systemically. The absence of atarget expression profile similarly favorable to CD19 thus prompted usto leverage the annotated database to explore combinatorial targetingstrategies.

Combinatorial Pairing of Candidate Targets

Combinatorial strategies fall in two major categories (FIG. 9). One isbased on cumulative CAR targeting through the generation of bi-specificT cells that co-express two CARs (or a dual-specific CAR (Duong et al.,2011; Grada et al., 2013; Wilkie et al., 2012; Zah et al., 2016). Theother takes advantage of split signaling (Alvarez-Vallina and Hawkins,1996; Krause et al., 1998) to target two antigens, using one antigen todirect costimulation to enhance or rescue the suboptimal function of aCAR or TCR targeting the other antigen (Kloss et al., 2013; Krause etal., 1998). In the former approach (CAR/CAR, FIG. 9), T cells recognizetarget cells that express any of two given antigens and will thus engagetissues expressing either antigen alone. Some low or moderate expressionin normal tissues, albeit not optimal, may be tolerable depending on thetissues in question. In the latter approach (CAR/CCR, FIG. 9), T cellsare more restricted to dual-antigen positive tumor cells, thus relaxingthe expression criteria for at least one of the paired antigens (FIG.9A). This approach however requires pan-tumor expression of the CARtarget to avert antigen escape (FIG. 9D). In both instances, targetpairings depend on the systemic expression and co-expression of the twoprospective matches to minimize cumulative expression in normal tissues.

To further improve the targeting of AML (approaching 100% FACSpositivity in all tumor cells as is seen with CD19 in ALL (Kong et al.,2008)), a software package was written to pair antigens minimizing thepotential for systemic on-target/off-tumor activity by avoidingcumulative target expression in normal organ/tissues (FIG. 9A). Twoother related safety requirements are the avoidance of normal HSC (FIG.9B) and T cell recognition (FIG. 9C). The considerations for acombinatorial approach are however more complex and entail additional,concurrent principles for selecting preferred target combinations. Thus,with regards to increasing therapeutic efficacy, the first principle isto maximize the number of targetable tumor cells, addressing thechallenge of clonal heterogeneity (FIG. 9D). Another priority is totarget LSCs, without which a CAR therapy would not stand a chance ofbeing potentially curative (FIG. 9E). Finally, pairing choices shouldfavor redundant expression of the two targets in the tumor in order tominimize the risk of antigen escape (FIG. 9F). These principles wereapplied to a pool of 12 molecules, including the 9 top single targetsdefined in FIG. 7, to which CD123, CD33 and CLEC12A were added, whichrepresent 66 possible combinations.

The first step was to identify antigen pairs that did not increasesystemic on-target/off-tumor tissue targeting, addressing the principleillustrated in FIG. 9A. To this end, a script that pairs targets withnon-overlapping expression in normal tissues were generated, whereinexpression levels in vital and non-vital tissues were weighted. Pairingin vital tissues required that at least one of two antigens be“not-detected” (0) in each tissue. Pairing in non-vital tissues allowedboth and one of the two antigens to exhibit “low” (1) expression. Ourtop 4 targets (ADGRE2, CCR1, CD70 and LILRB2), did not presentoverlapping expression in normal tissues (other than myeloid-richtissues-bone, blood, spleen, appendix) when paired with CD33, CLEC12A orCD96. Thus, several pairings appeared not to increase toxicity based onthis cumulative targeting criterion alone (FIG. 16). As CD96 was removedfrom final pairing because of its high expression in T cells (FIG. 8D)and failure to meet the FIG. 9C principle, the following 4 combinationswere further pursued for validation in primary AML samples: ADGRE2+CD33,CCR1⁺CLEC12A, CD70⁺CD33 and LILRB2+CLEC12A (FIG. 10A).

The pairing must also aim for maximum efficacy against AML and preventantigen escape, strive to recognize all AML cells in a given clinicalspecimen, prioritizing LSCs and favoring redundancy (FIG. 9D-F). Allfour combinations increased the rate of targeted targeting, reachingnearly 100% FACS positivity in all AML cells. For each one of thesepairs, the dual targeting exceeds the targeting of either antigen alone:(ADGRE2+CD33=97.5%) vs ADGRE2(93%) and CD33 (87.5%); (CLEC12A+CCR1=96%)vs CLEC12A (87.8%) and CCR1 (87%); (CD70+CD33=97.2%) vs CD70 (86%) andCD33 (87.5%); (LILRB2+CLEC12A=92.7%) vs LILRB2 (79.8%) (FIG. 10B).

The co-expression of two given targets (which would serve to preventantigen escape, principle FIG. 9F) were further analyzed in comparisonto the sum of each one's expression (which would serve to targetmultiple cancer clones, principle FIG. 9D). Most of the AML cellpopulations expressed both antigens in these best pairing (FIG. 10C). Itis however noteworthy that total positivity (union) was significantlyhigher than dual-positivity (intersection) (FIG. 10C), suggesting thepresence of minor clones expressing one antigen only. This finding isconsistent with clonal heterogeneity and favors using these antigenpairs in the dual-targeting approach (CAR/CAR, FIG. 9). A CAR/CCRcombinatorial targeting approach might in this instance increase therisk of relapse and antigen escape and disease relapse (FIG. 9).

Finally, expression levels of our 4 combinations in normal bone marrowHSPCs and peripheral blood T cells was low (FIG. 10D), confirming thatone can maximize AML recognition without increasing the risk of toxicitytowards normal hematopoietic cells.

Discussion

CAR therapy is a novel approach to cancer immunotherapy that hasdemonstrated great potential against relapsed B-cell malignancies, inparticular ALL. One may hope for a comparable outcome in AML, if targetsas effective as CD19 are identified. A platform that relies on largedata sets of protein and RNA expression in malignant and normal tissueswere generated, from which new candidate targets for AML CAR therapywere identified. However, neither these nor previously described CARtargets sufficiently fulfill the criteria for a suitable CAR target(Table 3). This led to instead employ the platform to identifycombinatorial pairings which can target nearly all AML cells within atumor sample, including LSCs, but without increasing off-target activityabove the level encountered with single targets. Here several targetpairs identified using this algorithm were reported, which exhibitnon-overlapping expression in normal tissues that would minimizesystemic on-target/off-tumor activity (FIGS. 9A and 10A), spare normalHSCs and T cells (FIG. 9B-C) and allow redundant targeting to nearly allAML cells, including LSCs with high redundancy, thereby addressing thechallenges of clonal heterogeneity and antigen escape (FIG. 9D-F and10B-C).

An extensive AML surfaceome dataset were assembled, combining publicprotein repositories and our own cell-surface proteomics performed in 6AML cell lines, thus generating an inclusive list of AML-associated cellsurface proteins. Studies on candidate targets typically focus on onemolecule, comparing expression in cancer cells to their normalcounterparts, but rarely do they take into account the systemicexpression of the candidate target, risking underestimation of thetoxicity across normal organs. To address body-wide protein expression,three extensive proteomics databases were combined, which map the humanproteome. Both immuno-histochemical assays and mass spectrometry datawere combined, increasing confidence especially for low levels ofexpression. This database included annotations of each candidatemolecule in a large panel of normal tissues, in addition to AML andnormal HSP cells. Notably, the inventors' integrated database confirmedthe presence of normal tissues adversely affected in earlier trialstargeting CAIX (Lamers et al., 2013; Lamers et al., 2006), CEA(Parkhurst et al., 2011) and ERBB2 (Morgan et al., 2010), includinggallbladder, gut and lung, respectively (FIG. 13). Conversely, CD19,whose only reported on-target/off-tumor toxicity is the induction of Bcell aplasia, exhibited a profile of expression limited to the expectedlymphoid-rich tissues (FIG. 7B).

Starting from over 5,000 Ensembl gene IDs (4,942 hgnc), the algorithmidentified 24 candidates with features potentially suitable for CARtargeting (FIG. 7). It should be noted, four of these targets, allpresent in our cell surface proteomics, were G-protein coupled receptors(G-PCRs): ADGRE2, CCR1, LTB4R and P2RY13. Prior studies based on RNA-seqhave found these G-PCRs to be amongst the most highly expressed G-PCRsin AML cells (Maiga et al., 2016). A possible role for G-PCRs inleukemic cell behavior has been suggested for chemokine receptors (suchas CCR1), adhesion receptors (such as ADGRE2) and purine receptors(including P2RY13) (Wilhelm et al., 2011). The FACS analyses conductedfor all 24 candidates in a panel of 30 primary AML samples and AML LSCs,further shortened our candidate list to nine molecules, based onpositive detection by FACS analysis in most patients and in >75% cellsper clinical specimen. Six of these molecules exhibited low levels ofexpression in normal bone marrow CD34+CD38− CD45RA-CD90+ HSCs: TNFRSF1B,ADGRE2, CCR1, CD96, CD70 and LILRB2. TNFRSF1B and CD96 were found to beexpressed at high levels in T cells (FIG. 8D), which may result in CAR Tcell self-elimination. The expression of TNFRSF1B and CD96 in T cellscould be eliminated by gene editing, however this would imposeadditional CAR T cell manufacturing steps (Riviere and Sadelain, 2017).

The remaining four candidate targets identified by our algorithm wereADGRE2, CCR1, CD70 and LILRB2. ADGRE2 (aka EMR2) is a member of theepidermal growth factor (EGF)-TM7 family of proteins, along with EMR1(Baud et al., 1995), F4/80 and CD97 (Lin et al., 1997). Like CD97,ADGRE2/EMR2 possesses calcium-binding EGF domains (Downing et al.,1996), but unlike CD97, which is ubiquitously expressed in many celltypes, EMR2 expression is restricted to monocytes/macrophages andgranulocytes and is not up-regulated in activated T and B cells (Lin etal., 2000)(FIG. 8E). ADGRE2 was found to be expressed at low levels inthe gut, ovary and spleen; conversely, its expression levels in AML werehigher compared to normal BM HSCs.

CCR1 (aka CD191) is a G-PCR that binds to members of the C-C chemokinefamily. An immunohistochemical analysis of 944 hematolymphoid neoplasiaspreviously identified CCR1 expression in a subset of AML, B and T celllymphomas, plasma cell myeloma, and Hodgkin lymphoma (Anderson et al.,2010). CCR1 presents the lowest overall expression in normal tissueswhile the majority of the AML cases showed strong CCR1 expression,averaging 88% positivity by FACS in all specimens.

CD70 is a member of the TNF-family and the ligand of the CD27 T cellcostimulatory receptor (Bowman et al., 1994; Goodwin et al., 1993). Itis expressed in multiple tumor types and serves as a target for antibodyand drug-conjugated antibody depletion in both renal cell carcinoma andnon-Hodgkin lymphoma (Law et al., 2006; McEarchern et al., 2007;McEarchern et al., 2008; Ryan et al., 2010). CD70-specific CARs havebeen shown to induce sustained regression of established Raji Burkittlymphoma xenografts (Shaffer et al., 2011). CD70 to be expressed at lowlevels in the gut and the FACS analyses detected CD70 in ˜86% of cellsin all patient specimens.

LILRB2 (aka CD85d) is a member of the leukocyte immunoglobulin-likereceptor (LIR) family. The encoded protein is expressed on myeloid and Bcells, acting to suppress the immune response. It is also expressed onNSCLC cells (Sun et al., 2008). LILRB2 to be expressed in thegallbladder and spleen at low levels and the FACS analyses detectedLILRB2 in ˜76% of cells in most patient specimens. This finding supportsthe notion that HSCs can express immune inhibitors of innate andadaptive immunity to evade potential immune surveillance (Zheng et al.,2012).

Seven targets have been previously reported as potential AML CARtargets. None of these meet our criteria for optimal single CARtargeting (Table 3), however they may nonetheless prove effective whileremaining within acceptable levels of toxicity. Expression profiles forthese seven targets in normal tissues are shown in FIG. 7B. CD33 is amyeloid-specific sialic acid-binding receptor, targeted by gentuzumabozogamicin (GO) (Administration, 2010) with demonstrated survivalbenefit in AML patients (Hills et al., 2014; Ravandi et al., 2012).Vadastuximab talirine, a CD33-directed antibody-drug conjugated in phaseIII clinical development (2016), was recently halted following 5 seriousadverse events in patients with AML. Preclinical studies evaluating CD33CARs have shown reduction of myeloid progenitors (Kenderian et al.,2015; Pizzitola et al., 2014). Two clinical trials targeting CD33 arecurrently active (NCT01864902 and NCT02799680). One AML patient wastreated with CD33 CAR T cells at the Chinese PLA General Hospital,showing transient efficacy and mild fluctuations in bilirubin (Wang etal., 2015). CD33 was found to be detected at higher levels than othermyeloid markers, as reflected in its higher expression in lung, skin,and prostate (FIG. 7B). The FACS analyses detected CD33 in ˜87% of cellsfrom all patient specimens. CLEC12A (aka CLL1, CD371), a type IItransmembrane receptor family containing a C-type lectin/C-typelectin-like domain, is over-expressed in LSCs (van Rhenen et al., 2007).The FACS analyses detected CLEC12A in the lung at low levels and in ˜87%of cells in most patient specimens. It is expressed in committedprogenitor cells, consistent with RNA expression levels (Bakker et al.,2004) and our flow cytometry results (FIG. 8C). CLEC12A plays a role asa negative regulator of granulocyte and monocyte function. CLEC12A CAR Tcells have been shown to be effective against HL60 (Tashiro et al.,2017), but exhibited modest activity against primary AML xenografts(Kenderian et al., 2016). Lewis (Le)-Y, a difucosylated carbohydrateantigen, has been targeted in four patients with relapsed AML. Infusionof second-generation CD28-based CAR T cells resulted in stable/transientremission of three patients, all of whom ultimately progressed, despiteT cell persistence (Ritchie et al., 2013), suggesting possible antigenescape. Le-Y was found to be highly expressed in the gut (FIG. 7) andthus did not consider it for further analysis. Two trials for CARstargeting CD123 (NCT02159495 and NCT02623582), the high-affinityinterleukin-3 receptor α-chain, are in progress. In one instance,partial remission was induced in a patient with FLT3-ITD+ AML treatedwith a third generation CD123-specific CAR (Luo et al., 2015).Preclinical studies however have revealed significant myeloablation inone study (Gill et al., 2014) but not another (Pizzitola et al., 2014).CD123 is expressed at high levels in several normal tissues (FIG. 7B),which resulted in its elimination by our algorithm. Low affinity CARsmay mitigate some of the on-target/off-tumor toxicity (Arcangeli et al.,2017). Moreover, folate receptor β and CD44v6, the isoform variant 6 ofthe adhesive receptor CD44, other myeloid-lineage antigens (Bendall etal., 2000; Legras et al., 1998; Lynn et al., 2016; Lynn et al., 2015)that were found to be expressed in multiple normal tissues (FIG. 7B).CD44v6 is found in AML stem cells (Casucci et al., 2013) and someepithelial tissues, particularly skin keratinocytes (Heider et al.,2004). Reports of CD44v6 expression are conflicting, depending onantibody usage (Bendall et al., 2000). CD38 is a non-lineage-restricted,type II transmembrane glycoprotein targeted by Daratumumab, the firstU.S. Food and Drug Administration-approved anti-CD38 antibody(Dimopoulos et al., 2016; Lonial et al., 2016) the activity of which onAML is limited without ATRA (Yoshida et al., 2016). It is expressed inall normal hematopoietic progenitor cells, T cell and NK cells. Morerecently, Drent et al. generated ˜124 antibodies specific for CD38spanning over 2 logs of affinity and demonstrated that CAR T cellsbearing scFvs with reduced affinity can strongly lyse CD38⁺⁺ myelomacells (on-target/on-tumor effect), while sparing CD38⁺ normalhematopoietic cells (on-target/off-tumor effect) (Drent et al., 2017).These findings extend previous reports showing a correlation betweenscFv affinity and CAR activity (Caruso et al., 2015; Hudecek et al.,2013; Liu et al., 2015).

While several of the above targets have therapeutic potential, none wasfound with an expression profile comparable favorable to CD19. Lesserabundant expression of these candidates in AML cells or LSCs as measuredby FACS analysis suggests a higher risk of antigen escape and AMLrelapse than seen with CD19 CAR therapy. These considerations promptedexploration of combinatorial targeting strategies. Combinatorialstrategies differ in both intent and approach (Sun and Sadelain, 2015;Wu et al., 2015). Some combine activating receptors (CAR/CAR), whichenables T cells to recognize target cells that express any of two givenantigens. This approach broadens T cell reactivity in the context of aheterogeneous disease like AML and likely decreases the risk of antigenescape, but at the cost of potentially accumulating toxicity associatedwith each target. In contrast, a combinatorial approach that restricts Tcell function to dual-positive tumor cells will avert T cell activityagainst normal tissues that express either target alone (Alvarez-Vallinaand Hawkins, 1996; Kloss et al., 2013), but requires pan-expression ofthe CAR target in AML cells. The analyses (FIG. 10B-C and FIG. 14),however, did not identify suitable CAR targets to implement thisstrategy.

6 principles (FIG. 9) were laid out to guide combinatorial CAR pairingwith the purpose of enhancing AML targeting (FIG. 9D-F, FIG. 10B-D)without increasing the potential for off-tumor toxicity (FIG. 9A-C and10A). From a pool of 12 promising molecules, pairwise combinationresults in 66 distinct pairs, from which several possible combinationswere suitable for further analysis. Four of these, CD33+ADGRE2,CLEC12A+CCR1, CD33+CD70 and LILRB2+CLEC12A, were studied in greaterdepth, by looking at their expression in primary AML specimens. Three ofthese pairings positively stained >97% of cells in AML samples,(LILRB2+CLEC12A scored slightly lower, averaging 93%, FIG. 10B), whileall stained <5% of normal HSCs and T cells. Thus, the aggregate stainingof ADGRE2 and CD33 increased the rate of FACS recognition to 97%. CD33is a relatively abundant myeloid marker, more abundant in lung, skin,and prostate than other myeloid markers, however this combinatorialpairing would not be expected to exacerbate on-target/off-tumor activity(FIG. 10A). Similarly, the combined targeting of CCR1 and CLEC12A is notpredicted to increase off-tumor targeting in the lung (FIG. 10A). Inpairing targets with non-overlapping expression in normal tissues, onemay leverage a co-targeting strategy with minimal cumulative antigenexpression in non-tumor cells (FIG. 9A).

This present study represents a new approach to the discovery of CARtargets and rests on two central concepts. First, the use of a compositehigh-throughput annotation database, including both proteomics andtranscriptomics, for evaluating many candidates simultaneously. Andsecond the application of six principles to guide combinatorialpairings, which are the basis for the algorithm applied here. Thisparadigm will help advance the development of CAR therapy for AML andother cancers including solid tumors.

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From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All patents and publications and sequences referred to by accession orreference number mentioned in this specification are herein incorporatedby reference to the same extent as if each independent patent andpublication and sequence was specifically and individually indicated tobe incorporated by reference.

What is claimed is:
 1. An immunoresponsive cell comprising: (a) anantigen recognizing receptor that binds to EMR2, wherein binding of theantigen recognizing receptor to EMR2 is capable of activating theimmunoresponsive cell; (b) a chimeric co-stimulating receptor (CCR) thatbinds to CLEC12A, wherein binding of the CCR to CLEC12A is capable ofstimulating the immunoresponsive cell, wherein the antigen recognizingreceptor binds to EMR2 with a binding affinity that is lower compared tothe binding affinity with which the CCR binds to CLEC12A.
 2. Apharmaceutical composition comprising an effective amount of animmunoresponsive cell of claim 1 and a pharmaceutically acceptableexcipient.
 3. A kit for treating and/or preventing a myeloid disorder,comprising an immunoresponsive cell of claim
 1. 4. The immunoresponsivecell of claim 1, wherein the antigen recognizing receptor is a chimericantigen receptor (CAR).
 5. The immunoresponsive cell of claim 4, whereinthe CAR comprises an intracellular signaling domain.
 6. Theimmunoresponsive cell of claim 5, wherein the intracellular signalingdomain of the CAR comprises a CD3ζ polypeptide.
 7. The immunoresponsivecell of claim 5, wherein the intracellular signaling domain of the CARcomprises at least one co-stimulatory signaling region.
 8. Theimmunoresponsive cell of claim 7, wherein the at least oneco-stimulatory signaling region comprises a signaling domain of CD28, asignaling domain of 4-1BB, a signaling domain of OX40, a signalingdomain of ICOS, a signaling domain of DAP-10, a signaling domain ofCD27, a signaling domain of CD154, a signaling domain of CD97, asignaling domain of Cd11a/CD18, a signaling domain of CD2, a signalingdomain of CD8, or a combination thereof.
 9. The immunoresponsive cell ofclaim 8, wherein the at least one co-stimulatory signaling regioncomprises a signaling domain of CD28.
 10. The immunoresponsive cell ofclaim 8, wherein the at least one co-stimulatory signaling regioncomprises a signaling domain of 4-1BB.
 11. The immunoresponsive cell ofclaim 4, wherein the CAR comprises a transmembrane domain.
 12. Theimmunoresponsive cell of claim 1, wherein the immunoresponsive cell isselected from the group consisting of an embryonic stem cell, apluripotent stem cell, a T cell, and a Natural Killer (NK) cell.
 13. Theimmunoresponsive cell of claim 1, wherein the immunoresponsive cell is aT cell.
 14. The immunoresponsive cell of claim 13, wherein the T cell isselected from the group consisting of a cytotoxic T cell (CTL), anatural killer T cell (NKT), a tumor infiltrating lymphocyte, and aregulatory T cell.
 15. The immunoresponsive cell of claim 1, wherein theimmunoresponsive cell is a natural killer (NK) cell.
 16. Theimmunoresponsive cell of claim 1, wherein the CCR does not alone deliveran activation signal to the cell.
 17. The immunoresponsive cell of claim1, wherein the CCR comprises an intracellular signaling domain.
 18. Theimmunoresponsive cell of claim 17, wherein the intracellular signalingdomain of the CCR does not comprise a CD3ζ polypeptide.
 19. Theimmunoresponsive cell of claim 17, wherein the intracellular signalingdomain of the CCR comprises a signaling domain of CD28, a signalingdomain of 4-1BB, a signaling domain of OX40, a signaling domain of ICOS,a signaling domain of DAP-10, or a combination thereof.
 20. Theimmunoresponsive cell of claim 1, wherein the antigen recognizingreceptor and/or the CCR is recombinantly expressed.
 21. Theimmunoresponsive cell of claim 1, wherein the antigen recognizingreceptor and/or the CCR is expressed from a vector.
 22. Theimmunoresponsive cell of claim 1, wherein the immunoresponsive cell isautologous.
 23. The immunoresponsive cell of claim 1, wherein theimmunoresponsive cell is allogenic.
 24. The immunoresponsive cell ofclaim 1, wherein the antigen receptor cell binds to EMR2 with adissociation constant (K_(d)) of 1×10⁻⁸ M or more, 5×10⁻⁸ M or more, or1×10⁻⁷ M or more.